Melampomagnolide B derivatives

ABSTRACT

The present disclosure provides derivatives of melampomagnolide B (MMB), including carbonates, carbamates, thiocarbamates, ester and amide derivatives of MMB. These derivatives are useful for treating cancer in humans.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/676,537, filed Apr.1, 2015, which is a continuation-in-part of U.S. non-provisionalapplication Ser. No. 14/537,389, filed Nov. 10, 2014, which claims thebenefit of U.S. provisional application No. 61/901,714, filed Nov. 8,2013, each of the disclosure of which are hereby incorporated byreference in their entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under Grant No. CA158275awarded by the National Institutes of Health (NIH). The government hascertain rights to the invention.

FIELD OF THE INVENTION

This disclosure generally relates to a series of melampomagnolide B(MMB) derivatives, including carbamate, thiocarbamate, carbonate, esterand amide derivatives of MMB. These compounds exhibit potent anticanceractivity.

BACKGROUND OF THE INVENTION

Parthenolide (PTL), an abundant sesquiterpene lactone found in themedicinal herb feverfew (Tanacetum parthenium), has undergone intensepharmacological research, especially for its antileukemic properties.Initial biomechanistic studies of PTL and its derivatives indicate thatthe compound promotes apoptosis by inhibiting the NF-kB transcriptionfactor complex, thereby downregulating antiapoptotic genes under NF-kBcontrol. PTL and its derivatives may also interfere with glutathionefunction, specifically glutathione's ability to sequester reactiveoxygen species. In culture, PTL induces robust apoptosis of primaryacute myeloid leukemia (AML) cells. To overcome poor water-solubility,PTL may be derivatized with an alkylamino, which can convert intowater-soluble salts. A series of fluorinated amino derivatives of PTLexhibit activity in antiproliferative assays in HL-60 (humanpromyelocytic leukemia) cells. PTL has also been the source of severalantileukemic compounds arising from chemical modification of the PTLmolecule.

Melampomagnolide B (MMB), a melampolide originally isolated fromMagnolia grandiflora, is an antileukemic sesquiterpene with propertiessimilar to those of PTL. MMB has been synthesized via selenium oxideoxidation of the C10 methyl group of PTL, resulting in a concomitantconversion of the geometry of the C9-C10 double bond from trans to thecis geometry. MMB contains a primary OH group, providing a point ofattachment for derivatives with increased water solubility,bioavailability, and tissue targeting. Phase 1 clinical data fromdimethylaminoparthenolide (DMAPT), a synthetic aminoparthenolidederivative, indicated improved bioavailability and longer in vivohalf-lifes for PTL and MMB derivatives with increased water solubility.

SUMMARY OF THE INVENTION

Briefly, therefore, one aspect of the present disclosure encompassescompounds comprising Formula (I):

wherein:

-   -   R is selected from the group consisting of

wherein:

-   -   R₁ is selected from the group consisting of substituted indoles,        substituted heterocyclic aromatic, aromatic and aliphatic        derivatives;    -   R₂ is selected from the group consisting of substituted indoles;        and    -   R₃ is selected from the group consisting of substituted amines.

Another aspect of the disclosure provides a process for preparing acompound comprising Formula (I). The process comprises (a) contacting acompound comprising MMB with a compound selected from the groupconsisting of: (i) a compound comprising a carboxylic acid; and (ii) acompound comprising an acyl chloride; (b) contacting a compoundcomprising melampomagnolic acid with a compound comprising aheterocyclic amine; or (c) contacting a compound comprising an aminederivative of MMB with a compound comprising a carboxylic acid to form acompound comprising Formula (I):

wherein:

-   -   R is selected from the group consisting of

wherein:

-   -   R₁ is selected from the group consisting of substituted indoles,        substituted heterocyclic aromatic, aromatic and aliphatic        derivatives;    -   R₂ is selected from the group consisting of substituted indoles;        and    -   R₃ is selected from the group consisting of substituted amines.

Yet another aspect of the disclosure provides a method for inhibitinggrowth of a cancer cell. The method comprises contacting the cancer cellwith an amount of a compound comprising Formula (I), or a salt thereof,effective to inhibit growth of the cancer cell.

Other features and iterations of the invention are described in moredetail below.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows the antileukemic activity of PTL, MMB, JMV 64, JMV 66, JMV69, and JMV 74 against the AML 052308 cell line as a function ofconcentration (μM) and percentage of cell viability.

FIG. 2 shows the antileukemic activity of PTL, MMB, JMV 57, JMV 58, JMV59, and JMV 61 against the AML 052308 cell line as a function ofconcentration (μM) and percentage of cell viability.

FIG. 3 shows the 24-hour M9's percentage of live cells relative tountreated cells for PTL, MMB, JMV 2-5, JMV 2-16, JMV 2-40, JMV 2-49, JVM2-35, JVM 2-41, and JVM 95 as a function of concentration (μM).

FIG. 4 shows the 24-hour M9's percentage of live cells relative tountreated cells for PTL, MMB, JMV 2-26, JMV 2-31, JMV 2-4, JMV 86, JVM90, and JVM 96 as a function of concentration (μM).

FIG. 5 shows the antileukemic activity of PTL, MMB, and JMV 88 againstthe M9 ENL cell line as a function of concentration (μM) and percentageof cell viability at 24 hours.

FIG. 6 shows the antileukemic activity of PTL, MMB, and JMV 88 againstthe AML 123009 cell line as a function of concentration (μM) andpercentage of cell viability.

FIG. 7 shows the antileukemic activity of PTL, MMB, and JMV 88 againstthe AML 100510 cell line as a function of concentration (μM) andpercentage of cell viability at 24 hours.

FIG. 8 shows structures of PTL (1), DMAPT fumarate (2) and MMB (3).

FIG. 9 shows the cytotoxic mechanism of action of MMB analogs.

FIG. 10 shows a graph demonstrating the anti-leukemic activity ofcarbamate and carbonate derivatives of melampomagnolide B against M9 ENLcells.

FIG. 11 shows a graph demonstrating the anti-leukemic activity ofsuccinic amide derivatives of melampomagnolide B against M9 ENL cells.

FIG. 12 shows a graph demonstrating the percentage of live cellsrelative to untreated cells after 24 hour-treatment with the compounds.Cells are M9 cells.

FIG. 13 shows a graph demonstrating the toxicity of PTL and BS-2-04against cord blood cells. BS-2-04 has an EC₅₀ of 0.72 μM in M9 cells andan EC₅₀ of 0.75 μM in cord blood cells. SI=1.04

FIG. 14 shows a graph demonstrating the percentage of live cellsrelative to untreated cells after 24 hour-treatment with the compounds.Cells are M9 cells.

FIG. 15 shows a graph demonstrating the cytotoxic activity of MMB esterconjugates against M9 cell lines.

FIG. 16 shows a graph demonstrating the cytotoxic activity of MMB amideconjugates against M9 cell lines.

FIG. 17A-I shows graphs demonstrating the five dose test results ofBS-1-28 against human cancer cell lines. (FIG. 17A) Leukemia; (FIG. 17B)Non-Small Cell Lung Cancer; (FIG. 17C) Colon Cancer; (FIG. 17D) CNSCancer; (FIG. 17E) Melanoma; (FIG. 17F) Ovarian Cancer; (FIG. 17G) RenalCancer; (FIG. 17H) Prostate Cancer; and (FIG. 17I) Breast Cancer.

FIG. 18 shows a graph demonstrating the five dose test results ofBS-1-28 against 60 human cancer cell lines.

FIG. 19A-B shows NCI single dose results for compound JVM 4-14. FIG. 19Ashows leukemia, NSCLC, colon cancer and CNS cancer and FIG. 19B showsmelanoma, ovarian cancer, renal cancer, prostate cancer and breastcancer.

FIG. 20A-B shows NCI single dose results for compound JVM 4-19. FIG. 20Ashows leukemia, NSCLC, colon cancer and CNS cancer and FIG. 20B showsmelanoma, ovarian cancer, renal cancer, prostate cancer and breastcancer.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are carbamate, thiocarbamate, and carbonate conjugatesof MMB, which may be synthesized via an intermediate prepared byreacting MMB with carbonylditriazole to afford MMB triazole (JVM 2-16,Example 10). This triazole intermediate may be reacted with variousheterocyclic amines, including, for example, imidazole, morpholine,piperidine, pyrrolidine, triazole, and pyridine, to afford thecorresponding carbamate conjugate. To prepare carbonate conjugates ofMMB, MMB triazole may be reacted with hydroxyl-containing compounds,including methanol, ethanol, N,N-dimethylethanolamine,morpholinoethanol, and piperidinopropanol. A thiocarbamate conjugate(JVM-66, Example 6) may be synthesized by reacting MMB withthiocarbonyldiimidazole. Also provided herein are carbamate andcarbonate conjugates of MMB, which may be synthesized via anintermediate prepared by reacting MMB with p-nitrophenylchloroformate toafford an ester of MMB. This ester derivative may be reacted withvarious heterocyclic amines, including, for example, imidazole,morpholine, piperidine, pyrrolidine, triazole, and pyridine, orhydroxyl-containing compounds, including methanol, ethanol,N,N-dimethylethanolamine, morpholinoethanol, and piperidinopropanol toafford the corresponding carbamate or carbonate conjugate.

Also provided herein are a series of novel ester and amide conjugates ofMMB. Various heterocyclic carboxylate conjugates of MMB may be preparedfrom substituted indole, benzothiophene, benzofuran, nicotinic,thiophene, aromatic and aliphatic carboxylic acids. MMB may be reactedwith various heterocyclic carboxylic acids in the presence of standardEDCI coupling conditions to furnish the respective MMB conjugates.Additionally, a variety of substituted heterocyclic aromatic andaliphatic amide conjugates of MMB were prepared by reaction of theappropriate amine with melampomagnolic acid, an oxidation product ofMMB, or the appropriate carboxylic acid with an amine derivative of MMB.Melampomagnolic acid may be reacted with various heterocyclic aminesunder the same standard coupling conditions affording the correspondingMMB amide conjugates. Alternatively, an amine derivative of MMB may bereacted with various heterocyclic carboxylic acids under the samestandard coupling conditions affording the corresponding MMB amideconjugates. Further, MMB may be reacted with various substitutedheterocyclic 2-(1H-indo-3-yl)-2-oxoacetyl chlorides in the presence oftriethylamine to afford the corresponding MMB ester conjugates.

These compounds were tested for anticancer activity against primary andnon-primary AML cell lines and various solid tumor cell lines. Severalcompounds were efficient anticancer agents against the AML cell linesand various solid tumor cell lines. Notably, the compounds weresignificantly more potent than the parent compounds PTL and MMB.

In general, the compounds detailed herein include compounds comprising amelampomagnolide B (MMB) structure as diagrammed below. For the purposesof illustration, the ring atoms of MMB are numbered as shown below:

MMB compounds have asymmetric centers. In particular, the MMB compoundsmay have at least four chiral carbons (designated by asterisks in thediagram above); namely, C4, C5, C6, and C7.

I. Compounds Comprising Formulas (I), (II), (III), (IV), (V), (VI),(VII) or (VIII)

In an aspect, provided herein are compounds comprising Formula (I):

wherein:

-   -   R is selected from the group consisting of

wherein:

-   -   R₁, R₂ and R₃ are independently selected from the group        consisting of —NR₆R₇, —OR₆, —O-alkyl-NR₆R₇, —SR₆,        —S-alkyl-NR₆R₇, alkyl-C(O)NR₆R₇, and -alkyl-R₈, substituted        indoles, substituted heterocyclic aromatic, aromatic and        aliphatic derivatives, and substituted amines;    -   R₆ is selected from the group consisting of hydrocarbyl,        substituted hydrocarbyl, and R₈;    -   R₇ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl;    -   R₈ is an optionally substituted nitrogen-containing heterocyclic        ring;    -   one or more of R₆ and R₇ may form part of a ring or ring system        chosen from the group consisting of heterocyclic, substituted        heterocyclic, and combinations thereof; and    -   when R₇ is hydrogen, R₆ is selected from the group consisting of        alkyl, R₈, and substituted hydrocarbyl having at least one        hydroxyl or R₈.

In some embodiments, R₁, R₂ and R₃ are selected from the groupconsisting of alkoxy, alkylamino, dialkylamino, dialkylaminoalkoxy,heterocyclylalkoxy, hydroxyalkylamino, heterocyclylamino, andheterocyclylalkylamino. In some embodiments, R₁, R₂ and R₃ may beselected from the group consisting of methylamino, dimethylamino,hydroxyhexylamino, hydroxyethylamino, pyrrolyl, pyrrolidinyl, pyridinyl,piperdinyl, pyrazinyl, piperazinyl, pyrimidinyl, imidazolyl, triazolyl,hydroxypiperdinyl, difluoropiperdinyl, triazolylamino,methylthiotriazolylamino, morpholinyl, morpholinylethylamino,pyridinylmethylamino, piperdinylethylamino, pyridinylethylamino,morpholinylpropylamino, imidiazolylpropylamino, methoxy,dimethylaminoethoxy, piperdinylpropoxy, piperdinylethoxy,pyrrolidinylethoxy, morpholinylethyoxy,piperidinylethoxyhydroxyethylthio, and piperdinylethyl. In otherembodiments, R₁, R₂ and R₃ may be selected from the group consisting ofimidazolyl-propylaminocarbonylethylcarbonyl,difluoropiperinylcarbonylethylcarbonyl,methylthio-triazolylaminocarbonylethylcarbonyl,chloropyridinylmethylaminocarbonylethylcarbonyl,methylpiperdinylcarbonylethylcarbonyl, andmethylpiperazinylcarbonylethylcarbonyl.

In certain embodiments, R₁ is selected from the group consisting ofsubstituted indoles, substituted heterocyclic aromatic, aromatic andaliphatic derivatives.

In certain embodiments, R₂ is selected from the group consisting ofsubstituted indoles.

In certain embodiments, R₃ is selected from the group consisting ofsubstituted amines.

In another aspect, provided herein are a compound comprising Formula(II) or (III):

wherein:

-   -   X is O or S;    -   R₄ and R₅ are independently selected from the group consisting        of —NR₆R₇, —OR₆, —O-alkyl-NR₆R₇, —SR₆, —S-alkyl-NR₆R₇,        alkyl-C(O)NR₆R₇, and -alkyl-R₈;    -   R₆ is selected from the group consisting of hydrocarbyl,        substituted hydrocarbyl, and R₈;    -   R₇ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl;    -   R₈ is an optionally substituted nitrogen-containing heterocyclic        ring;    -   one or more of R₆ and R₇ may form part of a ring or ring system        chosen from the group consisting of heterocyclic, substituted        heterocyclic, and combinations thereof; and    -   when R₇ is hydrogen, R₆ is selected from the group consisting of        alkyl, R₈, and substituted hydrocarbyl having at least one        hydroxyl or R₈.

In an exemplary embodiment of a compound comprising Formula (II), X isO; R₄ is NR₆R₇; and R₆, R₇ and R₈ are as described above.

In some embodiments, R₄ and R₅ may be selected from the group consistingof alkoxy, alkylamino, dialkylamino, dialkylaminoalkoxy,heterocyclylalkoxy, hydroxyalkylamino, heterocyclylamino, andheterocyclylalkylamino. In some exemplary embodiments, R₄ and R₅ may beselected from the group consisting of methylamino, dimethylamino,hydroxyhexylamino, hydroxyethylamino, pyrrolyl, pyrrolidinyl, pyridinyl,piperdinyl, pyrazinyl, piperazinyl, pyrimidinyl, imidazolyl, triazolyl,hydroxypiperdinyl, difluoropiperdinyl, triazolylamino,methylthiotriazolylamino, morpholinyl, morpholinylethylamino,pyridinylmethylamino, piperdinylethylamino, pyridinylethylamino,morpholinylpropylamino, imidiazolylpropylamino, methoxy, dimethylaminoethoxy, piperdinylpropoxy, piperdinylethoxy,pyrrolidinylethoxy, morpholinylethyoxy,piperidinylethoxyhydroxyethylthio, and piperdinylethyl. In otherexemplary embodiments, R₄ and R₅ may be independently selected from thegroup consisting of imidazolyl-propylaminocarbonylethylcarbonyl,difluoropiperinylcarbonylethylcarbonyl,methylthio-triazolylaminocarbonylethylcarbonyl,chloropyridinylmethylaminocarbonylethylcarbonyl,methylpiperdinylcarbonylethylcarbonyl, andmethylpiperazinylcarbonylethylcarbonyl. In a particular embodiment, R₄may be R₈, and R₅ may be dialkylamino. In still other embodiments, oneor more R₄, R₅, R₆, R₇, or R₈ may be substituted with at least oneselected from the group consisting of methyl, ethyl, propyl, cyano,C₁-C₃-alkylamino, carboxyl, hydroxyl, trifluoromethyl, thio, alkylthio,and halogen.

In another aspect, provided herein are compounds comprising Formula(IV):

wherein:

-   -   X is selected from the group consisting of S, O, NH, NR₁₀;    -   R₉ is selected from the group consisting of OMe, Cl, Br and F;        and    -   R₁₀ is selected from the group consisting of H, CH₃, benzyl,        substituted benzyl, benzoyl, substituted benzoyl, and        benzylsulfonyl and substituted benzylsulfonyl.

In still another aspect, provided herein are compounds comprisingFormula (V):

wherein:

-   -   R₁₁ is selected from the group consisting of H, CH₃, benzyl,        substituted benzyl, benzoyl, substituted benzoyl, and        benzylsulfonyl and substituted benzylsulfonyl; and    -   R₁₂ and R₁₃ are independently selected from the group consisting        of H, F, Cl, Br, OCH₃, CN, CH₃, NO₂ and COOCH₃.

In still another aspect, provided herein are compounds comprisingFormula (VI):

wherein:

-   -   R₁₄ is selected from the group consisting of 2-thiophenyl,        3-thiophenyl, 2-pyrazine, 3-pyrazine, 2-amino nicotinic acid,        indole-3-acetic acid, indole-3-acrylic acid.

In still another aspect, provided herein are compounds comprisingFormula (VII):

wherein:

-   -   R₁₅ is selected from the group consisting of:

In yet still another aspect, provided herein are compounds comprisingFormula (VIII):

wherein:

-   -   R₁ is selected from the group consisting of R₁ is selected from        the group consisting of substituted indoles, substituted        heterocyclic aromatic, aromatic and aliphatic derivatives.        (a) Downstream Applications

In some embodiments, the compound comprising Formula (I), (II), (III),(IV), (V), (VI), (VII) or (VIII) may be converted into apharmaceutically acceptable salt. “Pharmaceutically acceptable salts”are salts commonly used to form alkali metal salts and to form additionsalts of free acids or free bases. The nature of the salt may vary,provided that it is pharmaceutically acceptable.

Suitable pharmaceutically acceptable acid addition salts of compoundscomprising Formula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII)may be prepared from an inorganic acid or from an organic acid. Examplesof such inorganic acids are hydrochloric, hydrobromic, hydroiodic,nitric, carbonic, sulfuric and phosphoric acid. Appropriate organicacids may be selected from aliphatic, cycloaliphatic, aromatic,aliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which are oxalic, formic, acetic, propionic,succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic,anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, algenic, hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable pharmaceutically acceptablebase addition salts include metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine-(N-methylglucamine) andprocaine. All of these salts may be prepared by conventional means fromthe corresponding compound by reacting, for example, the appropriateacid or base with the any compound comprising Formula (I), (II), (III),(IV), (V), (VI), (VII) or (VIII).

(b) Stereochemistry

The compounds comprising Formula (I), (II), (III), (IV), (V(VI), (VII)or (VIII) may independently have an optical activity of (−) or (+). Inparticular, the configuration of C4, C5, C6, and C7, respectively, maybe RRRR, RRSR, RRRS, RRSS, RSRR, RSSR, RSRS, RSSS, SRRR, SRSR, SRRS,SRSS, SSRR, SSSR, SSRS, or SSSS.

II. Processes for Preparing Compounds Comprising Formula (I), (II),(III), (IV), (V), (VI), (VII) or (VIII)

In particular, provided herein are processes for preparing a compoundcomprising Formula (II) or (III). In general, the process comprisescontacting MMB with an appropriate reagent to form a compound comprisingFormula (II) or (III).

In an aspect, provided herein are processes for preparing a compoundcomprising Formula (II) or (III). The process comprises (a) contactingMMB with a triazole reagent to form a triazole intermediate. The processcontinues with (b) contacting the triazole intermediate with a compoundcomprising formula R⁴—H to form a compound comprising Formula (II) or(III).

In another aspect, provided herein are processes for preparing acarbamate or carbonate compound comprising Formula (II). The processcomprises (a) contacting MMB with p-nitrophenylchloroformate to form anester derivative of MMB. The process continues with (b) contacting theester derivative of MMB with a compound comprising formula R⁴—H to forma compound comprising Formula (II).

In still another aspect, provided herein are processes for preparing anamide compound comprising Formula (II). The process comprises (a)contacting MMB with an acid anhydride to form a carboxylic acidderivative. The process continues with (b) contacting the carboxylicacid derivative with a compound comprising formula R⁴—H to form acompound comprising Formula (II).

In still yet another aspect, provided herein are processes for preparinga compound comprising Formula (I), (II), (IV) or (VI). The processgenerally comprises (a) contacting MMB with a carboxylic acid to form acompound comprising Formula (I), (II), (IV) or (VI). Optionally, priorto step (a), an ester may be converted into the corresponding carboxylicacid prior to conjugation with MMB.

In a different aspect, provided herein are processes for preparing acompound comprising Formula (I), (II), (V) or (VI). The processgenerally comprises (a) contacting MMB with an acyl chloride to form acompound comprising Formula (I), (II), (V) or (VI). Optionally, prior tostep (a), a compound may be converted into the corresponding acylchloride prior to conjugation with MMB.

In certain aspects, provided herein are processes for preparing acompound comprising Formula (I) or (VII). The process generallycomprises (a) contacting melampomagnolic acid with a heterocyclic amineto form a compound comprising Formula (I) or (VII).

In other aspects, provided herein are processes for preparing a compoundcomprising Formula (I) or (VIII). The process generally comprises (a)contacting an amine derivative of MMB with a carboxylic acid to form acompound comprising Formula (I) or (VIII).

(a) Carbamate and Carbonate Derivatives of MMB Via a TriazoleIntermediate

Carbamate derivatives may be synthesized using, for example, phosgene,acid chlorides, carbamoyl chloride, or 1,1-carbonyldiimidazole (CDI). Insome embodiments, CDI may form a carbamate ester on MMB. CDIadvantageously provides easy handling, low expense, and relatively lowtoxicity. In particular embodiments, imidazole carboxylic esters may beformed by reaction of CDI with MMB. In this reaction imidazole, while abyproduct, also participated in an unexpected Michael addition reactionwith the MMB exocyclic double bond of the carbamate product (Scheme 1).

Thiocarbamate derivatives of MMB may be formed by reacting MMB withthiocarbonyl diimidazole dissolved in dichloromethane. If the reactionis maintained for 3-4 h, the major product was the Michael adduct JVM66A. If the reaction is run for a shorter time (e.g., 1 hour), thethiocarbamate (JVM 66) was the major product (Scheme 2). A detailedsynthesis of JVM 66 is provided below at Example 6.

The triazole derivative of MMB (JVM 2-16) was prepared by reacting MMBwith carbonylditriazole (CTD) dissolved in dichloromethane. The triazolebyproduct does not participate in a Michael addition reaction becausetriazole has only weak nucleophilic properties. The triazoleintermediate (JVM 2-16) reacted with various heterocyclic amines andalcohols to form a variety of carbamate and carbonate derivatives(Scheme 3). A detailed synthesis of JVM 2-16 is provided below atExample 10.

In still other embodiments, the triazole intermediate (JVM 2-16) may bereacted with mercaptoethanol in the presence of triethylamine to affordJVM 2-41. A detailed synthesis of JVM 2-41 is provided below at Example15.

In particular, provided herein are processes for preparing a compoundcomprising Formula (II) or (III). The process comprises (a) contactingMMB with a triazole reagent to form a triazole intermediate. The processcontinues with (b) contacting the triazole intermediate with a compoundcomprising formula R⁴—H to form a compound comprising Formula (II) or(III). This process is illustrated according to the following reactionscheme:

wherein:

-   -   X is O or S;    -   R₄ is selected from the group consisting of —NR₆R₇, —OR₆,        —O-alkyl-NR₆R₇, —SR₆, —S-alkyl-NR₆R₇, alkyl-C(O)NR₆R₇, and        -alkyl-R₈;    -   R₆ is selected from the group consisting of hydrocarbyl,        substituted hydrocarbyl, and R₈;    -   R₇ is selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl;    -   R₈ is an optionally substituted nitrogen-containing heterocyclic        ring;    -   one or more of R₆ and R₇ may form part of a ring or ring system        chosen from the group consisting of heterocyclic, substituted        heterocyclic, and combinations thereof; and    -   when R₇ is hydrogen, R₆ is selected from the group consisting of        alkyl, R₈, and substituted hydrocarbyl having at least one        hydroxyl or R₈.        (i) Step (a)—Reaction Mixture

Step (a) of the process comprises contacting MMB with a triazole reagentto form a triazole intermediate. The process commences with theformation of a reaction mixture comprising MMB, which is detailed above,the triazole reagent, and optionally a solvent system.

The triazole reagent may be any compound which reacts with a hydroxylgroup to provide a triazolyl carbamate. Non-limiting examples ofsuitable triazole reagents include carbonylditriazole (such as1,1′-carbonyl-di-(1,2,4-triazole) and thiocarbonylditriazole. Anotherpossible synthetic approach to the synthesis of the imidazole carbamateanalog of MMB is by utilizing carbonyldiimidazole as a reagent insteadof carbonylditriazole.

The amounts of triazole reagent that are contacted with MMB may vary. Ingeneral, the mole to mole ratio of MMB to triazole agent may range fromabout 1:0.2 to about 1:15. In certain embodiments, the mole to moleratio of MMB to the triazole reagent may range from about 1:0.2 to about1:0.7, from about 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5,from about 1:2.5 to about 1:5, from about 1:5 to about 1:10, or fromabout 1:10 to about 1:15. In certain embodiments, the mole to mole ratioof MMB to the triazole reagent may range from about 1:0.7 to about 1:3.

The reaction is generally conducted in the presence of a solvent orsolvent system. The solvent may be a polar aprotic solvent, a polarprotic solvent, or a nonpolar solvent. Non-limiting examples of suitablepolar aprotic solvents include acetone, acetonitrile, diethoxymethane,N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),N,N-dimethylpropanamide (or dimethylpropionamide; DMP),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxyethane (DME),dimethoxymethane, bis(2-methoxyethyl)ether, N,N-dimethylacetamide (DMA),N-methyl-2-pyrrolidinone (NMP), 1,4-dioxane, ethyl formate, formamide,hexachloroacetone, hexamethylphosphoramide, methyl acetate,N-methylacetamide, N-methylformamide, methylene chloride(dichloromethane, DCM), chloroform, methoxyethane, morpholine,nitrobenzene, nitromethane, propionitrile, pyridine, sulfolane,tetramethylurea, tetrahydrofuran (THF), 2-methyl tetrahydrofuran,tetrahydropyran, trichloromethane, and combinations thereof.Non-limiting examples of suitable polar protic solvents include water;alcohols such as methanol, ethanol, isopropanol, n-propanol, isobutanol,n-butanol, s-butanol, t-butanol, and the like; diols such as propyleneglycol; organic acids such as formic acid, acetic acid, and so forth;amides such as formamide, acetamide, and the like; and combinations ofany of the above. Representative nonpolar solvents include, but are notlimited to, alkane and substituted alkane solvents (includingcycloalkanes), aromatic hydrocarbons, esters, ethers, ketones, andcombinations thereof. Specific polar aprotic solvents that may beemployed include, for example, dichloromethane, chloroform, andcombinations thereof.

In general, the volume to mass ratio of the solvent to MMB ranges fromabout 1:1 to about 100:1. In various embodiments, the volume to massratio of the solvent to MMB may range from about 1:1 to about 5:1, fromabout 5:1 to about 10:1, from about 10:1 to about 20:1, from about 20:1to about 30:1, from about 30:1 to about 40:1, from about 40:1 to about50:1, from about 50:1 to about 60:1, from about 60:1 to about 70:1, fromabout 70:1 to about 80:1, from about 80:1 to about 90:1, or from about90:1 to about 100:1. In exemplary embodiments, the volume to mass ratioof the solvent to MMB may range from about 20:1 to about 30:1.

(ii) Step (a)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. In this context, a“completed reaction” generally means that the reaction mixture containsa significantly diminished amount MMB, and a significantly increasedamount of the triazole intermediate compared to the amounts of eachpresent at the beginning of the reaction. Typically, the amount MMBremaining in the reaction mixture after the reaction is complete may beless than about 3%, or less than about 1%. In general, the reaction mayproceed for about 5 minutes to about 48 hours. Typically, the durationof the reaction is longer at lower reaction temperatures. In certainembodiments, the reaction may be allowed to proceed for about a periodof time ranging from about 5 minutes to about 10 minutes, from about 10minutes to about 15 minutes, from about 15 minutes to about 30 minutes,from about 30 minutes to about 1 hour, about 1 hour to about 3 hours,from about 3 hours to about 6 hours, from about 6 hours to about 12hours, from about 12 hours to about 18 hours, from about 18 hours toabout 24 hours, from about 24 hours to about 36 hours, or from about 36hours to about 48 hours. In certain embodiments, the reaction may beallowed to proceed about 5 minutes to about 15 minutes. In otherembodiments, the reaction may be allowed to proceed about 45 minutes toabout 75 minutes. In still other embodiments, the reaction may beallowed to proceed about 18 hours to about 36 hours.

Generally, the triazole intermediate is not isolated and step (b) of theprocess proceeds in the same reaction pot or reactor. In someembodiments, the triazole intermediate may be isolated from the reactionmixture using techniques known to those of skill in the art.Non-limiting examples of suitable techniques include precipitation,extraction, evaporation, distillation, chromatography, andcrystallization.

The yield of the triazole intermediate can and will vary. Typically, theyield of the triazole intermediate may be at least about 40%. In oneembodiment, the yield of the triazole intermediate may range from about40% to about 60%. In another embodiment, the yield of the triazoleintermediate may range from about 60% to about 80%. In a furtherembodiment, the yield of the triazole intermediate may range from about80% to about 90%. In still another embodiment, the yield of the triazoleintermediate may be greater than about 90%, or greater than about 95%.

(iii) Step (b)—Reaction Mixture

Step (b) of the process continues with (b) contacting the triazoleintermediate with a compound comprising formula R⁴—H to form a compoundcomprising Formula (I) or (II). The process commences with the formationof a reaction mixture comprising the triazole intermediate, which isdetailed above, a compound comprising the formula R⁴—H, and optionally asolvent system.

In some embodiments, the compound comprising formula R⁴—H may beselected from the group consisting of imidazole, benzimidazole,morpholine, piperidine, pyrrole, pyrrolidine, triazole, tetrazole,piperazine, pyridine, pyrazoloimidazole, methanol, ethanol,N,N-dimethylethanolamine, morpholinoethanol, and piperidinopropanol.

The amounts of the compound comprising formula R⁴—H that are contactedwith the triazole intermediate may vary. In general, the mole to moleratio of the triazole intermediate to the compound comprising formulaR⁴—H may range from about 1:0.2 to about 1:15. In certain embodiments,the mole to mole ratio of the triazole intermediate to the compoundcomprising formula R⁴—H may range from about 1:0.2 to about 1:0.7, fromabout 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5, from about1:2.5 to about 1:5, from about 1:5 to about 1:10, or from about 1:10 toabout 1:15. In certain embodiments, the mole to mole ratio of thetriazole intermediate to the compound comprising formula R⁴—H may rangefrom about 1:0.7 to about 1:3.

Contact with the compound comprising formula R⁴—H generally is conductedin the presence of a solvent or solvent system. Suitable solvents aredetailed above in Section II(a)(i). In exemplary embodiments, thesolvent may be dichloromethane, chloroform, or combinations thereof. Ingeneral, the volume to mass ratio of the solvent to the triazoleintermediate ranges from about 1:1 to about 100:1. In variousembodiments, the volume to mass ratio of the solvent to the triazoleintermediate may range from about 1:1 to about 5:1, from about 5:1 toabout 10:1, from about 10:1 to about 20:1, from about 20:1 to about30:1, from about 30:1 to about 40:1, from about 40:1 to about 50:1, fromabout 50:1 to about 60:1, from about 60:1 to about 70:1, from about 70:1to about 80:1, from about 80:1 to about 90:1, or from about 90:1 toabout 100:1. In exemplary embodiments, the volume to mass ratio of thesolvent to the triazole intermediate may range from about 20:1 to about30:1.

(iv) Step (b)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. Typically, the amount ofthe triazole intermediate remaining in the reaction mixture after thereaction is complete may be less than about 3%, or less than about 1%.In general, the reaction may proceed for about 5 minutes to about 48hours. Typically, the duration of the reaction is longer at lowerreaction temperatures. In certain embodiments, the reaction may beallowed to proceed for about a period of time ranging from about 5minutes to about 10 minutes, from about 10 minutes to about 15 minutes,from about 15 minutes to about 30 minutes, from about 30 minutes toabout 1 hour, about 1 hour to about 3 hours, from about 3 hours to about6 hours, from about 6 hours to about 12 hours, from about 12 hours toabout 18 hours, from about 18 hours to about 24 hours, from about 24hours to about 36 hours, or from about 36 hours to about 48 hours. Incertain embodiments, the reaction may be allowed to proceed about 20minutes to about 40 minutes. In other embodiments, the reaction may beallowed to proceed about 8 hours to about 12 hours.

The yield of the compound comprising Formula (II) or (III) can and willvary. Typically, the yield of the compound comprising Formula (II) or(III) may be at least about 40%. In one embodiment, the yield of thecompound comprising Formula (II) or (III) may range from about 40% toabout 60%. In another embodiment, the yield of the compound comprisingFormula (II) or (III) may range from about 60% to about 80%. In afurther embodiment, the yield of the compound comprising Formula (II) or(III) may range from about 80% to about 90%. In still anotherembodiment, the yield of the compound comprising Formula (II) or (III)may be greater than about 90%, or greater than about 95%.

(b) Carbamate and Carbonate Derivatives of MMB Via an Ester Derivative

In still yet other embodiments, MMB (3) may be reacted withp-nitrophenylchloroformate (4) in the presence of triethylamine to formthe p-nitrophenyloxycarbonyl ester of MMB (5). Thep-nitrophenyloxycarbonyl ester of MMB may then be reacted with variousprimary and secondary heterocyclic amines to afford carbamate analogs ofMMB (6) (Scheme 4).

In particular, provided herein are processes for preparing a carbamateor carbonate compound comprising Formula (II) or (III). The processcomprises (a) contacting MMB (3) with p-nitrophenylchloroformate to forman ester derivative of MMB (e.g. (5)). The process continues with (b)contacting the ester derivative of MMB with a compound comprisingformula R⁴—H to form a compound comprising Formula (II) or (III).

(i) Step (a)—Reaction Mixture

Step (a) of the process comprises contacting MMB withp-nitrophenylchloroformate to form an ester derivative of MMB (e.g.(5)). The process commences with the formation of a reaction mixturecomprising MMB, which is detailed above, p-nitrophenylchloroformate, andoptionally a solvent system.

The amounts of p-nitrophenylchloroformate that are contacted with MMBmay vary. In general, the mole to mole ratio of MMB top-nitrophenylchloroformate may range from about 1:0.2 to about 1:15. Incertain embodiments, the mole to mole ratio of MMB top-nitrophenylchloroformate may range from about 1:0.2 to about 1:0.7,from about 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5, fromabout 1:2.5 to about 1:5, from about 1:5 to about 1:10, or from about1:10 to about 1:15. In certain embodiments, the mole to mole ratio ofMMB to p-nitrophenylchloroformate may range from about 1:0.7 to about1:3. In an exemplary embodiment, the mole to mole ratio of MMB top-nitrophenylchloroformate may be about 1:0.7.

The reaction is generally conducted in the presence of a solvent orsolvent system. The solvent may be a polar aprotic solvent, a polarprotic solvent, or a nonpolar solvent. Non-limiting examples of suitablepolar aprotic solvents include acetone, acetonitrile, diethoxymethane,N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),N,N-dimethylpropanamide (or dimethylpropionamide; DMP),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxyethane (DME),dimethoxymethane, bis(2-methoxyethyl)ether, N,N-dimethylacetamide (DMA),N-methyl-2-pyrrolidinone (NMP), 1,4-dioxane, ethyl formate, formamide,hexachloroacetone, hexamethylphosphoramide, methyl acetate,N-methylacetamide, N-methylformamide, methylene chloride(dichloromethane, DCM), chloroform, methoxyethane, morpholine,nitrobenzene, nitromethane, propionitrile, pyridine, sulfolane,tetramethylurea, tetrahydrofuran (THF), 2-methyl tetrahydrofuran,tetrahydropyran, trichloromethane, and combinations thereof.Non-limiting examples of suitable polar protic solvents include water;alcohols such as methanol, ethanol, isopropanol, n-propanol, isobutanol,n-butanol, s-butanol, t-butanol, and the like; diols such as propyleneglycol; organic acids such as formic acid, acetic acid, and so forth;amides such as formamide, acetamide, and the like; and combinations ofany of the above. Representative nonpolar solvents include, but are notlimited to, alkane and substituted alkane solvents (includingcycloalkanes), aromatic hydrocarbons, esters, ethers, ketones, andcombinations thereof. Specific polar aprotic solvents that may beemployed include, for example, dichloromethane, chloroform, andcombinations thereof.

A proton acceptor is generally added to facilitate the reaction. Theproton acceptor generally has a pKa greater than about 7, or from about7 to about 13, or more specifically from about 9 to about 11.Representative proton acceptors may include, but are not limited to,borate salts (such as, for example, NaBO₃), di- and tri-basic phosphatesalts, (such as, for example, Na₂HPO₄ and NaPO₄), bicarbonate salts,carbonate salts, hydroxides, alkoxides, (including methoxide, ethoxide,propoxide, butoxide, and pentoxide, including straight chain andbranched), and organic proton acceptors, (such as, for example,pyridine, triethylamine, N-methylmorpholine, andN,N-dimethylaminopyridine), and mixtures thereof. In some embodiments,the proton acceptor may be stabilized by a suitable counterion such aslithium, potassium, sodium, calcium, magnesium, and the like. In aspecific embodiment, the proton acceptor is triethylamine. The amount ofproton acceptor included in the reaction can and will vary, but can bereadily determined by a person of ordinary skill in the art.

In general, the volume to mass ratio of the solvent to MMB ranges fromabout 1:1 to about 100:1. In various embodiments, the volume to massratio of the solvent to MMB may range from about 1:1 to about 5:1, fromabout 5:1 to about 10:1, from about 10:1 to about 20:1, from about 20:1to about 30:1, from about 30:1 to about 40:1, from about 40:1 to about50:1, from about 50:1 to about 60:1, from about 60:1 to about 70:1, fromabout 70:1 to about 80:1, from about 80:1 to about 90:1, or from about90:1 to about 100:1. In exemplary embodiments, the volume to mass ratioof the solvent to MMB may range from about 20:1 to about 30:1. In otherexemplary embodiments, the volume to mass ratio of the solvent to MMBmay range from about 10:1 to about 20:1.

(ii) Step (a)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. In this context, a“completed reaction” generally means that the reaction mixture containsa significantly diminished amount of MMB, and a significantly increasedamount of the carboxylic acid derivative compared to the amounts of eachpresent at the beginning of the reaction. Typically, the amount of MMBremaining in the reaction mixture after the reaction is complete may beless than about 3%, or less than about 1%. In general, the reaction mayproceed for about 5 minutes to about 48 hours. Typically, the durationof the reaction is longer at lower reaction temperatures. In certainembodiments, the reaction may be allowed to proceed for about a periodof time ranging from about 5 minutes to about 10 minutes, from about 10minutes to about 15 minutes, from about 15 minutes to about 30 minutes,from about 30 minutes to about 1 hour, about 1 hour to about 3 hours,from about 3 hours to about 6 hours, from about 6 hours to about 12hours, from about 12 hours to about 18 hours, from about 18 hours toabout 24 hours, from about 24 hours to about 36 hours, or from about 36hours to about 48 hours. In certain embodiments, the reaction may beallowed to proceed about 5 minutes to about 15 minutes. In otherembodiments, the reaction may be allowed to proceed about 45 minutes toabout 75 minutes. In still other embodiments, the reaction may beallowed to proceed about 18 hours to about 36 hours. In an exemplaryembodiment, the reaction may be allowed to proceed about 24 hours.

Generally, the ester derivative is not isolated and step (b) of theprocess proceeds in the same reaction pot or reactor. In someembodiments, the ester derivative may be isolated from the reactionmixture using techniques known to those of skill in the art.Non-limiting examples of suitable techniques include precipitation,extraction, evaporation, distillation, chromatography, andcrystallization.

The yield of the ester derivative can and will vary. Typically, theyield of the ester derivative may be at least about 40%. In oneembodiment, the yield of the ester derivative may range from about 40%to about 60%. In another embodiment, the yield of the ester derivativemay range from about 60% to about 80%. In a further embodiment, theyield of the ester derivative may range from about 80% to about 90%. Instill another embodiment, the yield of the ester derivative may begreater than about 90%, or greater than about 95%.

(iii) Step (b)—Reaction Mixture

Step (b) of the process continues with (b) contacting the esterderivative with a compound comprising formula R⁴—H to form a compoundcomprising Formula (II) or (III). The process commences with theformation of a reaction mixture comprising the ester derivative, whichis detailed above, a compound comprising the formula R⁴—H, andoptionally a solvent system.

In some embodiments, the compound comprising formula R⁴—H may beselected from the group consisting of imidazole, benzimidazole,morpholine, piperidine, pyrrole, pyrrolidine, triazole, tetrazole,piperazine, pyridine, pyrazoloimidazole, methanol, ethanol,N,N-dimethylethanolamine, morpholinoethanol, and piperidinopropanol.

The amounts of the compound comprising formula R⁴—H that are contactedwith the ester derivative may vary. In general, the mole to mole ratioof the ester derivative to the compound comprising formula R⁴—H mayrange from about 1:0.2 to about 1:15. In certain embodiments, the moleto mole ratio of the ester derivative to the compound comprising formulaR⁴—H may range from about 1:0.2 to about 1:0.7, from about 1:0.7 toabout 1:1.5, from about 1:1.5 to about 1:2.5, from about 1:2.5 to about1:5, from about 1:5 to about 1:10, or from about 1:10 to about 1:15. Incertain embodiments, the mole to mole ratio of the ester derivative tothe compound comprising formula R¹—H may range from about 1:0.7 to about1:3. In an exemplary, the mole to mole ratio of the ester derivative tothe compound comprising formula R⁴—H may be about 1:1.

Contact with the compound comprising formula R⁴—H generally is conductedin the presence of a solvent or solvent system. Suitable solvents aredetailed above in Section II(b)(i). In exemplary embodiments, thesolvent may be dichloromethane, chloroform, or combinations thereof. Ingeneral, the volume to mass ratio of the solvent to the ester derivativeranges from about 1:1 to about 100:1. In various embodiments, the volumeto mass ratio of the solvent to the ester derivative may range fromabout 1:1 to about 5:1, from about 5:1 to about 10:1, from about 10:1 toabout 20:1, from about 20:1 to about 30:1, from about 30:1 to about40:1, from about 40:1 to about 50:1, from about 50:1 to about 60:1, fromabout 60:1 to about 70:1, from about 70:1 to about 80:1, from about 80:1to about 90:1, or from about 90:1 to about 100:1. In exemplaryembodiments, the volume to mass ratio of the solvent to the esterderivative may range from about 20:1 to about 30:1. In another exemplaryembodiment, the volume to mass ratio of the solvent to the esterderivative may range from about 10:1 to about 20:1.

(iv) Step (b)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. Typically, the amount ofthe ester derivative remaining in the reaction mixture after thereaction is complete may be less than about 3%, or less than about 1%.In general, the reaction may proceed for about 5 minutes to about 48hours. Typically, the duration of the reaction is longer at lowerreaction temperatures. In certain embodiments, the reaction may beallowed to proceed for about a period of time ranging from about 5minutes to about 10 minutes, from about 10 minutes to about 15 minutes,from about 15 minutes to about 30 minutes, from about 30 minutes toabout 1 hour, about 1 hour to about 3 hours, from about 3 hours to about6 hours, from about 6 hours to about 12 hours, from about 12 hours toabout 18 hours, from about 18 hours to about 24 hours, from about 24hours to about 36 hours, or from about 36 hours to about 48 hours. Incertain embodiments, the reaction may be allowed to proceed about 20minutes to about 40 minutes. In other embodiments, the reaction may beallowed to proceed about 5 hours to about 18 hours. In exemplaryembodiments, the reaction may be allowed to proceed about 5 hours toabout 12 hours.

The yield of the compound comprising Formula (II) or (III) can and willvary. Typically, the yield of the compound comprising Formula (II) or(III) may be at least about 40%. In one embodiment, the yield of thecompound comprising Formula (II) or (III) may range from about 40% toabout 60%. In another embodiment, the yield of the compound comprisingFormula (II) or (III) may range from about 60% to about 80%. In afurther embodiment, the yield of the compound comprising Formula (II) or(III) may range from about 80% to about 90%. In still anotherembodiment, the yield of the compound comprising Formula (II) or (III)may be greater than about 90%, or greater than about 95%.

(c) Amide Derivatives of MMB Via an Acid Anhydride

In other embodiments, MMB may be reacted with succinic anhydride inpresence of triethylamine to afford a carboxylic acid derivative of MMB,JVM 67. The MMB carboxylic acid derivative may be reacted withheterocyclic amines to afford the corresponding amide derivatives of MMB(Scheme 5). A detailed synthesis of JVM 67 is provided below at Example7.

In particular, provided herein are processes for preparing an amidecompound comprising Formula (II) or (III). The process comprises (a)contacting MMB with an acid anhydride to form a carboxylic acidderivative. The process continues with (b) contacting the carboxylicacid derivative with a compound comprising formula R⁴—H to form acompound comprising Formula (II) or (III).

(i) Step (a)—Reaction Mixture

Step (a) of the process comprises contacting MMB with an acid anhydrideto form a carboxylic acid derivative. The process commences with theformation of a reaction mixture comprising MMB, which is detailed above,the acid anhydride, and optionally a solvent system.

The acid anhydride may be any compound which reacts with a hydroxylgroup to provide a carboxylic acid derivative. A suitable acid anhydrideis a compound that has two acyl groups bonded to the same oxygen atom.In a preferred embodiment, the acid anhydride is a cyclic anhydride.Non-limiting examples of suitable acid anhydrides include succinicanhydride, maleic anhydride, itaconic anhydride, citraconic anhydrideand 2-pentenedioic anhydride. In an exemplary embodiment, the acidanhydride is succinic anhydride.

The amounts of acid anhydride that are contacted with MMB may vary. Ingeneral, the mole to mole ratio of MMB to acid anhydride may range fromabout 1:0.2 to about 1:15. In certain embodiments, the mole to moleratio of MMB to acid anhydride may range from about 1:0.2 to about1:0.7, from about 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5,from about 1:2.5 to about 1:5, from about 1:5 to about 1:10, or fromabout 1:10 to about 1:15. In certain embodiments, the mole to mole ratioof MMB to acid anhydride may range from about 1:0.7 to about 1:3. In anexemplary embodiment, the mole to mole ratio of MMB to acid anhydridemay range from about 1:1.

The reaction is generally conducted in the presence of a solvent orsolvent system. The solvent may be a polar aprotic solvent, a polarprotic solvent, or a nonpolar solvent. Non-limiting examples of suitablepolar aprotic solvents include acetone, acetonitrile, diethoxymethane,N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),N,N-dimethylpropanamide (or dimethylpropionamide; DMP),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxyethane (DME),dimethoxymethane, bis(2-methoxyethyl)ether, N,N-dimethylacetamide (DMA),N-methyl-2-pyrrolidinone (NMP), 1,4-dioxane, ethyl formate, formamide,hexachloroacetone, hexamethylphosphoramide, methyl acetate,N-methylacetamide, N-methylformamide, methylene chloride(dichloromethane, DCM), chloroform, methoxyethane, morpholine,nitrobenzene, nitromethane, propionitrile, pyridine, sulfolane,tetramethylurea, tetrahydrofuran (THF), 2-methyl tetrahydrofuran,tetrahydropyran, trichloromethane, and combinations thereof.Non-limiting examples of suitable polar protic solvents include water;alcohols such as methanol, ethanol, isopropanol, n-propanol, isobutanol,n-butanol, s-butanol, t-butanol, and the like; diols such as propyleneglycol; organic acids such as formic acid, acetic acid, and so forth;amides such as formamide, acetamide, and the like; and combinations ofany of the above. Representative nonpolar solvents include, but are notlimited to, alkane and substituted alkane solvents (includingcycloalkanes), aromatic hydrocarbons, esters, ethers, ketones, andcombinations thereof. Specific polar aprotic solvents that may beemployed include, for example, dichloromethane, chloroform, andcombinations thereof.

A proton acceptor is generally added to facilitate the reaction. Theproton acceptor generally has a pKa greater than about 7, or from about7 to about 13, or more specifically from about 9 to about 11.Representative proton acceptors may include, but are not limited to,borate salts (such as, for example, NaBO₃), di- and tri-basic phosphatesalts, (such as, for example, Na₂HPO₄ and NaPO₄), bicarbonate salts,carbonate salts, hydroxides, alkoxides, (including methoxide, ethoxide,propoxide, butoxide, and pentoxide, including straight chain andbranched), and organic proton acceptors, (such as, for example,pyridine, triethylamine, N-methylmorpholine, andN,N-dimethylaminopyridine), and mixtures thereof. In some embodiments,the proton acceptor may be stabilized by a suitable counterion such aslithium, potassium, sodium, calcium, magnesium, and the like. In aspecific embodiment, the proton acceptor is triethylamine. The amount ofproton acceptor included in the reaction can and will vary, but can bereadily determined by a person of ordinary skill in the art.

In general, the volume to mass ratio of the solvent to MMB ranges fromabout 1:1 to about 100:1. In various embodiments, the volume to massratio of the solvent to MMB may range from about 1:1 to about 5:1, fromabout 5:1 to about 10:1, from about 10:1 to about 20:1, from about 20:1to about 30:1, from about 30:1 to about 40:1, from about 40:1 to about50:1, from about 50:1 to about 60:1, from about 60:1 to about 70:1, fromabout 70:1 to about 80:1, from about 80:1 to about 90:1, or from about90:1 to about 100:1. In exemplary embodiments, the volume to mass ratioof the solvent to MMB may range from about 20:1 to about 30:1. In otherexemplary embodiments, the volume to mass ratio of the solvent to MMBmay range from about 10:1 to about 20:1.

(ii) Step (a)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. In this context, a“completed reaction” generally means that the reaction mixture containsa significantly diminished amount of MMB, and a significantly increasedamount of the carboxylic acid derivative compared to the amounts of eachpresent at the beginning of the reaction. Typically, the amount of MMBremaining in the reaction mixture after the reaction is complete may beless than about 3%, or less than about 1%. In general, the reaction mayproceed for about 5 minutes to about 48 hours. Typically, the durationof the reaction is longer at lower reaction temperatures. In certainembodiments, the reaction may be allowed to proceed for about a periodof time ranging from about 5 minutes to about 10 minutes, from about 10minutes to about 15 minutes, from about 15 minutes to about 30 minutes,from about 30 minutes to about 1 hour, about 1 hour to about 3 hours,from about 3 hours to about 6 hours, from about 6 hours to about 12hours, from about 12 hours to about 18 hours, from about 18 hours toabout 24 hours, from about 24 hours to about 36 hours, or from about 36hours to about 48 hours. In certain embodiments, the reaction may beallowed to proceed about 5 minutes to about 15 minutes. In otherembodiments, the reaction may be allowed to proceed about 45 minutes toabout 75 minutes. In still other embodiments, the reaction may beallowed to proceed about 36 hours to about 48 hours.

Generally, the carboxylic acid derivative is not isolated and step (b)of the process proceeds in the same reaction pot or reactor. In someembodiments, the carboxylic acid derivative may be isolated from thereaction mixture using techniques known to those of skill in the art.Non-limiting examples of suitable techniques include precipitation,extraction, evaporation, distillation, chromatography, andcrystallization.

The yield of the carboxylic acid derivative can and will vary.Typically, the yield of the carboxylic acid derivative may be at leastabout 40%. In one embodiment, the yield of the carboxylic acidderivative may range from about 40% to about 60%. In another embodiment,the yield of the carboxylic acid derivative may range from about 60% toabout 80%. In a further embodiment, the yield of the carboxylic acidderivative may range from about 80% to about 90%. In still anotherembodiment, the yield of the carboxylic acid derivative may be greaterthan about 90%, or greater than about 95%.

(iii) Step (b)—Reaction Mixture

Step (b) of the process continues with (b) contacting the carboxylicacid derivative with a compound comprising formula R⁴—H to form acompound comprising Formula (II) or (III). The process commences withthe formation of a reaction mixture comprising the carboxylic acidderivative, which is detailed above, a compound comprising the formulaR⁴—H, and optionally a solvent system.

In some embodiments, the compound comprising formula R⁴—H may beselected from the group consisting of imidazole, benzimidazole,morpholine, piperidine, pyrrole, pyrrolidine, triazole, tetrazole,piperazine, pyridine, pyrazoloimidazole, methanol, ethanol,N,N-dimethylethanolamine, morpholinoethanol, and piperidinopropanol.

The amounts of the compound comprising formula R⁴—H that are contactedwith the carboxylic acid derivative may vary. In general, the mole tomole ratio of the carboxylic acid derivative to the compound comprisingformula R⁴—H may range from about 1:0.2 to about 1:15. In certainembodiments, the mole to mole ratio of the carboxylic acid derivative tothe compound comprising formula R¹—H may range from about 1:0.2 to about1:0.7, from about 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5,from about 1:2.5 to about 1:5, from about 1:5 to about 1:10, or fromabout 1:10 to about 1:15. In certain embodiments, the mole to mole ratioof the carboxylic acid derivative to the compound comprising formulaR⁴—H may range from about 1:0.7 to about 1:3.

Contact with the compound comprising formula R⁴—H generally is conductedin the presence of a solvent or solvent system. Suitable solvents aredetailed above in Section II(c)(i). In exemplary embodiments, thesolvent may be dichloromethane, chloroform, or combinations thereof.Additionally, a proton acceptor is generally added to facilitate thereaction. Suitable proton acceptors are detailed above in SectionII(c)(i). In a specific embodiment, the proton acceptor istriethylamine. Further, peptide coupling agents may also be added to thereaction. Non-limiting examples of suitable peptide coupling agentsinclude EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)), HOBt(Hydroxybenzotriazole), DCC (N,N′-Dicyclohexylcarbodiimide), HATU((1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate)), HBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumHexafluorophosphate), and TBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate).In an exemplary embodiment, EDC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)) and HOBt(Hydroxybenzotriazole) are added to the reaction.

In general, the volume to mass ratio of the solvent to the carboxylicacid derivative ranges from about 1:1 to about 100:1. In variousembodiments, the volume to mass ratio of the solvent to the carboxylicacid derivative may range from about 1:1 to about 5:1, from about 5:1 toabout 10:1, from about 10:1 to about 20:1, from about 20:1 to about30:1, from about 30:1 to about 40:1, from about 40:1 to about 50:1, fromabout 50:1 to about 60:1, from about 60:1 to about 70:1, from about 70:1to about 80:1, from about 80:1 to about 90:1, or from about 90:1 toabout 100:1. In exemplary embodiments, the volume to mass ratio of thesolvent to the carboxylic acid derivative may range from about 20:1 toabout 30:1. In another exemplary embodiment, the volume to mass ratio ofthe solvent to the carboxylic acid derivative may range from about 10:1to about 20:1.

(iv) Step (b)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. In other embodiments, the reaction may be conducted at a temperaturefrom about 0° C. to about 25° C. The reaction generally is conducted inan inert atmosphere (e.g., under nitrogen or argon) and under ambientpressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. Typically, the amount ofthe carboxylic acid derivative remaining in the reaction mixture afterthe reaction is complete may be less than about 3%, or less than about1%. In general, the reaction may proceed for about 5 minutes to about 48hours. Typically, the duration of the reaction is longer at lowerreaction temperatures. In certain embodiments, the reaction may beallowed to proceed for about a period of time ranging from about 5minutes to about 10 minutes, from about 10 minutes to about 15 minutes,from about 15 minutes to about 30 minutes, from about 30 minutes toabout 1 hour, about 1 hour to about 3 hours, from about 3 hours to about6 hours, from about 6 hours to about 12 hours, from about 12 hours toabout 18 hours, from about 18 hours to about 24 hours, from about 24hours to about 36 hours, or from about 36 hours to about 48 hours. Incertain embodiments, the reaction may be allowed to proceed about 3hours to about 16 hours.

The yield of the compound comprising Formula (II) or (III) can and willvary. Typically, the yield of the compound comprising Formula (II) or(III) may be at least about 40%. In one embodiment, the yield of thecompound comprising Formula (II) or (III) may range from about 40% toabout 60%. In another embodiment, the yield of the compound comprisingFormula (II) or (III) may range from about 60% to about 80%. In afurther embodiment, the yield of the compound comprising Formula (II) or(III) may range from about 80% to about 90%. In still anotherembodiment, the yield of the compound comprising Formula (II) or (III)may be greater than about 90%, or greater than about 95%.

(d) Ester Derivatives of MMB Via Carboxylic Acids

Ester derivatives of MMB may be synthesized from the reaction of variousorganic carboxylic acids with MMB in the presence of EDCI(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) in dichloromethane. Thereaction may occur at about room temperature for about 8 hours tofurnish the corresponding MMB ester conjugate (Scheme 6).

In one embodiment, the carboxylic acid utilized in Scheme 6 may resultin the synthesis of simple and substituted indole, benzothiophene, andbenzofuran carboxylate ester conjugates of MMB. For example, thecarboxylic acid may be 5-methoxyindole-2-carboxylic acid,5-chloro-indole-2-carboxylic acid, 5-fluorindole-2-carboxylic acid,indole-3-acetic acid, indole-3-acrylic acid, indomethacin,benzothiophene-2-carboxylic acid, 3-chlorobenzothiopene-2-carboxylicacid, benzofuran-2-carboxylic acid. Specifically, these organic acidsmay be converted into their corresponding MMB ester conjugates: BS-2-01,2-30, 2-68, 2-32, 2-04, 2-71, 2-65, 2-64, 2-63 (below).

In another embodiment, the carboxylic acid utilized in Scheme 6 mayresult in the synthesis of MMB ester conjugates of various heterocycliccarboxylic acids. For example, the carboxylic acid may be2-aminonicotinic acid, pyrazine-2-carboxylic acid,5-methylthiophene-2-carboxylic acid, and acetic acid. Specifically,these organic acids may be converted into their corresponding MMB esterconjugates: BS-2-66, 2-67, 2-78, 2-59 (below).

In still another embodiment, the methyl esters of a carboxylic acid maybe treated with aqueous sodium hydroxide (NaOH) in methanol to affordthe corresponding carboxylic acid prior to conjugation with MMB. Forexample, the methyl esters of 5-indole carboxylic acid and 6-indolecarboxylic acid may be treated with aqueous NaOH in methanol to affordthe corresponding indole carboxylic acids prior to conjugation with MMButilizing the conditions of Scheme 6 to afford the respective MMB esterconjugates (Scheme 7).

In particular, provided herein are processes for preparing a compoundcomprising Formula (I), (II), (IV) or (VI). The process generallycomprises (a) contacting MMB with a carboxylic acid to form a compoundcomprising Formula (I), (II), (IV) or (VI). Optionally, prior to step(a), an ester may be converted into the corresponding carboxylic acidprior to conjugation with MMB.

(i) Step (a)—Reaction Mixture

Step (a) of the process comprises contacting MMB with a carboxylic acidto form an ester derivative of MMB. The process commences with theformation of a reaction mixture comprising MMB, a carboxylic acid, andoptionally a solvent system.

The carboxylic acid may be any compound which comprises a carboxylicacid group (—COOH). In certain embodiments, a carboxylic acid may besimple and substituted indole, benzothiophene, and benzofuran carboxylicacids. Non-limiting examples of simple and substituted indole,benzothiophene, and benzofuran carboxylic acids include5-methoxyindole-2-carboxylic acid, 5-chloro-indole-2-carboxylic acid,5-fluorindole-2-carboxylic acid, indole-3-acetic acid, indole-3-acrylicacid, indomethacin, benzothiophene-2-carboxylic acid,3-chlorobenzothiopene-2-carboxylic acid, and benzofuran-2-carboxylicacid. In other embodiments, a carboxylic acid may be variousheterocyclic carboxylic acids. Non-limiting examples of variousheterocyclic carboxylic acids include 2-aminonicotinic acid,pyrazine-2-carboxylic acid, 5-methylthiophene-2-carboxylic acid, aceticacid, 5-(2-oxohexahydro-1H-thienol[3,4-d]imidazol-4-yl)pentanoic acid,and12-(5-(2-oxohexahydro-1H-thieno[3,4-c]imidazol-4-yl)pentan-amido)dodecanoicacid.

The amounts of carboxylic acid that are contacted with MMB may vary. Ingeneral, the mole to mole ratio of MMB to carboxylic acid may range fromabout 1:0.2 to about 1:15. In certain embodiments, the mole to moleratio of MMB to carboxylic acid may range from about 1:0.2 to about1:0.7, from about 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5,from about 1:2.5 to about 1:5, from about 1:5 to about 1:10, or fromabout 1:10 to about 1:15. In certain embodiments, the mole to mole ratioof MMB to carboxylic acid may range from about 1:0.7 to about 1:3. In anexemplary embodiment, the mole to mole ratio of MMB to carboxylic acidmay be about 1:0.7.

The reaction is generally conducted in the presence of a solvent orsolvent system. The solvent may be a polar aprotic solvent, a polarprotic solvent, or a nonpolar solvent. Non-limiting examples of suitablepolar aprotic solvents include acetone, acetonitrile, diethoxymethane,N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),N,N-dimethylpropanamide (or dimethylpropionamide; DMP),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxyethane (DME),dimethoxymethane, bis(2-methoxyethyl)ether, N,N-dimethylacetamide (DMA),N-methyl-2-pyrrolidinone (NMP), 1,4-dioxane, ethyl formate, formamide,hexachloroacetone, hexamethylphosphoramide, methyl acetate,N-methylacetamide, N-methylformamide, methylene chloride(dichloromethane, DCM), chloroform, methoxyethane, morpholine,nitrobenzene, nitromethane, propionitrile, pyridine, sulfolane,tetramethylurea, tetrahydrofuran (THF), 2-methyl tetrahydrofuran,tetrahydropyran, trichloromethane, and combinations thereof.Non-limiting examples of suitable polar protic solvents include water;alcohols such as methanol, ethanol, isopropanol, n-propanol, isobutanol,n-butanol, s-butanol, t-butanol, and the like; diols such as propyleneglycol; organic acids such as formic acid, acetic acid, and so forth;amides such as formamide, acetamide, and the like; and combinations ofany of the above. Representative nonpolar solvents include, but are notlimited to, alkane and substituted alkane solvents (includingcycloalkanes), aromatic hydrocarbons, esters, ethers, ketones, andcombinations thereof. Specific polar aprotic solvents that may beemployed include, for example, dichloromethane, chloroform, andcombinations thereof.

Further, peptide coupling agents may also be added to the reaction. Apeptide coupling agent may be used as a carboxyl activating agent forthe coupling of primary amines to yield amide bonds. Non-limitingexamples of suitable peptide coupling agents include EDCI(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)), HOBt(Hydroxybenzotriazole), DCC (N,N′-Dicyclohexylcarbodiimide), HATU((1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate)), HBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumHexafluorophosphate), and TBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate).In an exemplary embodiment, EDCI(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)) is added to thereaction.

Additionally, a nucleophilic catalyst may be added to the reaction. Anucleophilic catalyst may be used for a variety of reactions such asesterifications with anhydrides, the Baylis-Hillman reaction,hydrosilylations, tritylation, the Steglich rearrangement, andStaudinger synthesis of β-lactams. Non-limiting examples of suitablenucleophilic catalysts include 4-Dimethylaminopyridine (DMAP) andN-Hydroxybenzotriazole (HOBt). In an exemplary embodiment DMAP is addedto the reaction.

In general, the volume to mass ratio of the solvent to MMB ranges fromabout 1:1 to about 100:1. In various embodiments, the volume to massratio of the solvent to MMB may range from about 1:1 to about 5:1, fromabout 5:1 to about 10:1, from about 10:1 to about 20:1, from about 20:1to about 30:1, from about 30:1 to about 40:1, from about 40:1 to about50:1, from about 50:1 to about 60:1, from about 60:1 to about 70:1, fromabout 70:1 to about 80:1, from about 80:1 to about 90:1, or from about90:1 to about 100:1. In exemplary embodiments, the volume to mass ratioof the solvent to MMB may range from about 20:1 to about 30:1. In otherexemplary embodiments, the volume to mass ratio of the solvent to MMBmay range from about 10:1 to about 20:1.

(ii) Step (a)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. In this context, a“completed reaction” generally means that the reaction mixture containsa significantly diminished amount of MMB, and a significantly increasedamount of the compound comprising Formula (I), (II), (IV) or (VI)compared to the amounts of each present at the beginning of thereaction. Typically, the amount of MMB remaining in the reaction mixtureafter the reaction is complete may be less than about 3%, or less thanabout 1%. In general, the reaction may proceed for about 5 minutes toabout 48 hours. Typically, the duration of the reaction is longer atlower reaction temperatures. In certain embodiments, the reaction may beallowed to proceed for about a period of time ranging from about 5minutes to about 10 minutes, from about 10 minutes to about 15 minutes,from about 15 minutes to about 30 minutes, from about 30 minutes toabout 1 hour, about 1 hour to about 3 hours, from about 3 hours to about6 hours, from about 6 hours to about 12 hours, from about 12 hours toabout 18 hours, from about 18 hours to about 24 hours, from about 24hours to about 36 hours, or from about 36 hours to about 48 hours. Incertain embodiments, the reaction may be allowed to proceed about 6hours to about 12 hours. In other embodiments, the reaction may beallowed to proceed about 6 hours to about 10 hours. In still otherembodiments, the reaction may be allowed to proceed about 8 hours.

The yield of the compound comprising Formula (I), (II), (IV) or (VI) canand will vary. Typically, the yield of the compound comprising Formula(I), (II), (IV) or (VI) may be at least about 40%. In one embodiment,the yield of the compound comprising Formula (I), (II), (IV) or (VI) mayrange from about 40% to about 60%. In another embodiment, the yield ofthe compound comprising Formula (I), (II), (IV) or (VI) may range fromabout 60% to about 80%. In a further embodiment, the yield of thecompound comprising Formula (I), (II), (IV) or (VI) may range from about80% to about 90%. In still another embodiment, the yield of the compoundcomprising Formula (I), (II), (IV) or (VI) may be greater than about90%, or greater than about 95%.

(iii) Optional Step

Optionally, prior to step (a), an ester is converted into thecorresponding carboxylic acid prior to conjugation with MMB. An estermay be any ester of a carboxylic acid described in Section II(d)(i)above. Methods to convert an ester to a carboxylic acid are known in theart. An aqueous acid such as H₂SO₄ or an aqueous base such as NaOH andheat may be used for the conversion. In certain embodiments, thisconversion may occur via reaction of a methyl ester with aqueous sodiumhydroxide (NaOH) in methanol to afford the corresponding carboxylicacid. In a specific embodiment, the methyl esters of 5-indole carboxylicacid and 6-indole carboxylic acid may be treated with aqueous NaOH inmethanol to afford the corresponding indole carboxylic acid.

(e) Ester Derivatives of MMB Via Chlorides

Ester derivatives of MMB may be synthesized from the reaction of variousacyl chlorides with MMB in the presence of triethylamine anddichloromethane. The reaction may occur at about room temperature forabout 1-18 hours to furnish the corresponding MMB ester conjugate(Scheme 8).

In one embodiment, ester conjugates may be synthesized by the reactionof MMB with substituted benzoyl and naphthoyl chlorides in the presenceof triethylamine as base and dichloromethane as solvent in ambienttemperature for 6-18 h. Specifically, these chlorides were convertedinto their corresponding MMB ester conjugates (below).

In an exemplary embodiment, MMB esters of the invention may be:

In another embodiment, analogues of indole carboxylate-MMB esterconjugates may be prepared by the reaction of MMB with a variety ofsubstituted 2-(1H-indol-3-yl)-2-oxoacetyl chlorides in the presence oftriethylamine at about room temperature for about 1 to 5 h. Thesubstituted 2-(1H-indol-3-yl)-2-oxoacetyl chlorides may be prepared bythe reaction of the appropriate indole with oxalyl chloride in diethylether at about 0° C. to about room temperature for about 1 h (Scheme 9).

In particular, provided herein are processes for preparing a compoundcomprising Formula (I), (II), (V) or (VI). The process generallycomprises (a) contacting MMB with an acyl chloride to form a compoundcomprising Formula (I), (II), (V) or (VI). Optionally, prior to step(a), a compound may be converted into the corresponding acyl chlorideprior to conjugation with MMB.

(i) Step (a)—Reaction Mixture

Step (a) of the process comprises contacting MMB with an acyl chlorideto form an ester derivative of MMB. The process commences with theformation of a reaction mixture comprising MMB, an acyl chloride, andoptionally a solvent system.

The acyl chloride may be any compound which comprises an acyl chloridegroup (—COCl). The compound comprising an acyl chloride may comprise analkyl chain. The alkyl chain may be linear, branched or contain anaromatic ring. The alkyl chain may be a hydrocarbyl alkyl chain or asubstituted hydrocarbyl alkyl chain. The alkyl chain may be a length of1 to 10 atoms. For example, the alkyl chain may be 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 atoms in length. In certain embodiments, an acyl chloride maybe a substituted benzoyl or napthoyl chloride. In other embodiments, anacyl chloride may be a variety of substituted2-(1H-indol-3-yl)-2-oxoacetyl chlorides.

The amounts of acyl chloride that are contacted with MMB may vary. Ingeneral, the mole to mole ratio of MMB to acyl chloride may range fromabout 1:0.2 to about 1:15. In certain embodiments, the mole to moleratio of MMB to acyl chloride may range from about 1:0.2 to about 1:0.7,from about 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5, fromabout 1:2.5 to about 1:5, from about 1:5 to about 1:10, or from about1:10 to about 1:15. In certain embodiments, the mole to mole ratio ofMMB to acyl chloride may range from about 1:0.7 to about 1:3. In anexemplary embodiment, the mole to mole ratio of MMB to acyl chloride maybe about 1:0.7.

The reaction is generally conducted in the presence of a solvent orsolvent system. The solvent may be a polar aprotic solvent, a polarprotic solvent, or a nonpolar solvent. Non-limiting examples of suitablepolar aprotic solvents include acetone, acetonitrile, diethoxymethane,N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),N,N-dimethylpropanamide (or dimethylpropionamide; DMP),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxyethane (DME),dimethoxymethane, bis(2-methoxyethyl)ether, N,N-dimethylacetamide (DMA),N-methyl-2-pyrrolidinone (NMP), 1,4-dioxane, ethyl formate, formamide,hexachloroacetone, hexamethylphosphoramide, methyl acetate,N-methylacetamide, N-methylformamide, methylene chloride(dichloromethane, DCM), chloroform, methoxyethane, morpholine,nitrobenzene, nitromethane, propionitrile, pyridine, sulfolane,tetramethylurea, tetrahydrofuran (THF), 2-methyl tetrahydrofuran,tetrahydropyran, trichloromethane, and combinations thereof.Non-limiting examples of suitable polar protic solvents include water;alcohols such as methanol, ethanol, isopropanol, n-propanol, isobutanol,n-butanol, s-butanol, t-butanol, and the like; diols such as propyleneglycol; organic acids such as formic acid, acetic acid, and so forth;amides such as formamide, acetamide, and the like; and combinations ofany of the above. Representative nonpolar solvents include, but are notlimited to, alkane and substituted alkane solvents (includingcycloalkanes), aromatic hydrocarbons, esters, ethers, ketones, andcombinations thereof. Specific polar aprotic solvents that may beemployed include, for example, dichloromethane, chloroform, andcombinations thereof.

A proton acceptor is generally added to facilitate the reaction. Theproton acceptor generally has a pKa greater than about 7, or from about7 to about 13, or more specifically from about 9 to about 11.Representative proton acceptors may include, but are not limited to,borate salts (such as, for example, NaBO₃), di- and tri-basic phosphatesalts, (such as, for example, Na₂HPO₄ and NaPO₄), bicarbonate salts,carbonate salts, hydroxides, alkoxides, (including methoxide, ethoxide,propoxide, butoxide, and pentoxide, including straight chain andbranched), and organic proton acceptors, (such as, for example,pyridine, triethylamine, N-methylmorpholine, andN,N-dimethylaminopyridine), and mixtures thereof. In some embodiments,the proton acceptor may be stabilized by a suitable counterion such aslithium, potassium, sodium, calcium, magnesium, and the like. In aspecific embodiment, the proton acceptor is triethylamine. The amount ofproton acceptor included in the reaction can and will vary, but can bereadily determined by a person of ordinary skill in the art.

In general, the volume to mass ratio of the solvent to MMB ranges fromabout 1:1 to about 100:1. In various embodiments, the volume to massratio of the solvent to MMB may range from about 1:1 to about 5:1, fromabout 5:1 to about 10:1, from about 10:1 to about 20:1, from about 20:1to about 30:1, from about 30:1 to about 40:1, from about 40:1 to about50:1, from about 50:1 to about 60:1, from about 60:1 to about 70:1, fromabout 70:1 to about 80:1, from about 80:1 to about 90:1, or from about90:1 to about 100:1. In exemplary embodiments, the volume to mass ratioof the solvent to MMB may range from about 20:1 to about 30:1. In otherexemplary embodiments, the volume to mass ratio of the solvent to MMBmay range from about 10:1 to about 20:1.

(ii) Step (a)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. In this context, a“completed reaction” generally means that the reaction mixture containsa significantly diminished amount of MMB, and a significantly increasedamount of the compound comprising Formula (I), (II), (V) or (VI)compared to the amounts of each present at the beginning of thereaction. Typically, the amount of MMB remaining in the reaction mixtureafter the reaction is complete may be less than about 3%, or less thanabout 1%. In general, the reaction may proceed for about 5 minutes toabout 48 hours. Typically, the duration of the reaction is longer atlower reaction temperatures. In certain embodiments, the reaction may beallowed to proceed for about a period of time ranging from about 5minutes to about 10 minutes, from about 10 minutes to about 15 minutes,from about 15 minutes to about 30 minutes, from about 30 minutes toabout 1 hour, about 1 hour to about 3 hours, from about 3 hours to about6 hours, from about 6 hours to about 12 hours, from about 12 hours toabout 18 hours, from about 18 hours to about 24 hours, from about 24hours to about 36 hours, or from about 36 hours to about 48 hours. Incertain embodiments, the reaction may be allowed to proceed about 1 hourto about 18 hours. In other embodiments, the reaction may be allowed toproceed about 1 hour to about 5 hours. In still other embodiments, thereaction may be allowed to proceed about 6 hours to about 18 hours.

The yield of the compound comprising Formula (I), (II), (V) or (VI) canand will vary. Typically, the yield of the compound comprising Formula(I), (II), (V) or (VI) may be at least about 40%. In one embodiment, theyield of the compound comprising Formula (I), (II), (V) or (VI) mayrange from about 40% to about 60%. In another embodiment, the yield ofthe compound comprising Formula (I), (II), (V) or (VI) may range fromabout 60% to about 80%. In a further embodiment, the yield of thecompound comprising Formula (I), (II), (V) or (VI) may range from about80% to about 90%. In still another embodiment, the yield of the compoundcomprising Formula (I), (II), (V) or (VI) may be greater than about 90%,or greater than about 95%.

(iii) Optional Step

Optionally, prior to step (a), a compound is converted into thecorresponding acyl chloride prior to conjugation with MMB. A compoundmay be the parent of an acyl chloride described in Section II(e)(i)above. A compound may be converted to an acyl chloride via reaction ofthe compound with oxalyl chloride (COCl)₂ in the presence of diethylether. The reaction may be carried out at about 0° C. to about 25° C.for about 1 hour. In a specific embodiment, various indoles may bereacted with oxalyl chloride in the presence of diethyl ether.

(f) Amide Derivatives of MMB

Amide derivatives of MMB may be synthesized from the reaction of variousheterocyclic amines with melampomagnolic acid in the presence of EDCI(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), hydroxybenzotriazole(HOBt) and triethylamine. The reaction may occur at about roomtemperature for about 3 to 8 hours to furnish the corresponding MMBamide conjugate (Scheme 10).

In an exemplary embodiment, MMB amides of the invention may be:

Alternatively, amide derivatives of MMB may be synthesized from thereaction of various aromatic or heteroaromatic carboxylic acids with anamine derivative of MMB in the presence of EDCI(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), hydroxybenzotriazole(HOBt) and triethylamine. The reaction may occur at about roomtemperature for about 3 to 15 hours to furnish the corresponding MMBamide conjugate (Scheme 11).

Scheme 11: Synthesis of Aromatic and Heteroaromatic Amide Derivatives ofMMB

In another exemplary embodiment, MMB amides of the invention may be:

In particular, provided herein are processes for preparing a compoundcomprising Formula (I) or (VII). The process generally comprises (a)contacting melampomagnolic acid with a heterocyclic amine to form acompound comprising Formula (I) or (VII).

Also, in particular, provided herein are processes for preparing acompound comprising Formula (I) or (VIII). The process generallycomprises (a) contacting an amine derivative of MMB with a carboxylicacid to form a compound comprising Formula (I) or (VIII).

(i) Step (a)—Reaction Mixture

Step (a) of the process comprises contacting melampomagnolic acid with aheterocyclic amine to form an amide derivative of MMB. The processcommences with the formation of a reaction mixture comprisingmelampomagnolic acid, a heterocyclic amine, and optionally a solventsystem. Alternatively, step (a) of the process comprises contacting anamine derivative of MMB with a carboxylic acid to form an amidederivative of MMB. The process commences with the formation of areaction mixture comprising an amine derivative of MMB, a carboxylicacid, and optionally a solvent system.

The heterocyclic amine may be any compound comprising a heterocycle andan amine group. The heterocyle may be an optionally substituted, fullysaturated or unsaturated, monocyclic or bicyclic, aromatic ornon-aromatic group having at least one heteroatom in at least one ring,and preferably 5 or 6 atoms in each ring. The heterocycle preferably has1 or 2 oxygen atoms and/or 1 to 4 nitrogen atoms in the ring.Non-limiting examples of suitable heterocyclic amines include imidazole,benzimidazole, morpholine, piperidine, pyrrole, pyrrolidine, triazole,tetrazole, piperazine, pyridine, pyrazoloimidazole, methanol, ethanol,N,N-dimethylethanolamine, morpholinoethanol, and piperidinopropanol.

The carboxylic acid may be any compound which comprises a carboxylicacid group (—COOH). In certain embodiments, the carboxylic acid may bean aromatic or heteroaromatic carboxylic acid. The aromatic orheteroaromatic ring may be an optionally substituted monocyclic,bicyclic, or tricyclic group containing from 5 to 14 atoms in the ringportion. For example, the aromatic or heteroaromatic ring may be aphenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl, orsubstituted naphthyl. In other embodiments, a carboxylic acid may besimple and substituted indole, benzothiophene, and benzofuran carboxylicacids. Non-limiting examples of simple and substituted indole,benzothiophene, and benzofuran carboxylic acids include5-methoxyindole-2-carboxylic acid, 5-chloro-indole-2-carboxylic acid,5-fluorindole-2-carboxylic acid, indole-3-acetic acid, indole-3-acrylicacid, indomethacin, benzothiophene-2-carboxylic acid,3-chlorobenzothiopene-2-carboxylic acid, and benzofuran-2-carboxylicacid. In other embodiments, a carboxylic acid may be variousheterocyclic carboxylic acids. Non-limiting examples of variousheterocyclic carboxylic acids include 2-aminonicotinic acid,pyrazine-2-carboxylic acid, 5-methylthiophene-2-carboxylic acid, aceticacid, 5-(2-oxohexahydro-1H-thienol[3,4-d]imidazol-4-yl)pentanoic acid,and12-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentan-amido)dodecanoicacid.

The amounts of heterocyclic amine or carboxylic acid that are contactedwith melampomagnolic acid or an amine derivative of MMB, respectively,may vary. In general, the mole to mole ratio of melampomagnolic acid toheterocyclic amine or amine derivative of MMB to carboxylic acid mayrange from about 1:0.2 to about 1:15. In certain embodiments, the moleto mole ratio of melampomagnolic acid to heterocyclic amine or aminederivative of MMB to carboxylic acid may range from about 1:0.2 to about1:0.7, from about 1:0.7 to about 1:1.5, from about 1:1.5 to about 1:2.5,from about 1:2.5 to about 1:5, from about 1:5 to about 1:10, or fromabout 1:10 to about 1:15. In certain embodiments, the mole to mole ratioof melampomagnolic acid to heterocyclic amine or amine derivative of MMBto carboxylic acid may range from about 1:0.2 to about 1:0.7. In anexemplary embodiment, the mole to mole ratio of melampomagnolic acid toheterocyclic amine or amine derivative of MMB to carboxylic acid may beabout 1:0.5.

The reaction is generally conducted in the presence of a solvent orsolvent system. The solvent may be a polar aprotic solvent, a polarprotic solvent, or a nonpolar solvent. Non-limiting examples of suitablepolar aprotic solvents include acetone, acetonitrile, diethoxymethane,N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),N,N-dimethylpropanamide (or dimethylpropionamide; DMP),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxyethane (DME),dimethoxymethane, bis(2-methoxyethyl)ether, N,N-dimethylacetamide (DMA),N-methyl-2-pyrrolidinone (NMP), 1,4-dioxane, ethyl formate, formamide,hexachloroacetone, hexamethylphosphoramide, methyl acetate,N-methylacetamide, N-methylformamide, methylene chloride(dichloromethane, DCM), chloroform, methoxyethane, morpholine,nitrobenzene, nitromethane, propionitrile, pyridine, sulfolane,tetramethylurea, tetrahydrofuran (THF), 2-methyl tetrahydrofuran,tetrahydropyran, trichloromethane, and combinations thereof.Non-limiting examples of suitable polar protic solvents include water;alcohols such as methanol, ethanol, isopropanol, n-propanol, isobutanol,n-butanol, s-butanol, t-butanol, and the like; diols such as propyleneglycol; organic acids such as formic acid, acetic acid, and so forth;amides such as formamide, acetamide, and the like; and combinations ofany of the above. Representative nonpolar solvents include, but are notlimited to, alkane and substituted alkane solvents (includingcycloalkanes), aromatic hydrocarbons, esters, ethers, ketones, andcombinations thereof. Specific polar aprotic solvents that may beemployed include, for example, dichloromethane, chloroform, andcombinations thereof.

A proton acceptor is generally added to facilitate the reaction. Theproton acceptor generally has a pKa greater than about 7, or from about7 to about 13, or more specifically from about 9 to about 11.Representative proton acceptors may include, but are not limited to,borate salts (such as, for example, NaBO₃), di- and tri-basic phosphatesalts, (such as, for example, Na₂HPO₄ and NaPO₄), bicarbonate salts,carbonate salts, hydroxides, alkoxides, (including methoxide, ethoxide,propoxide, butoxide, and pentoxide, including straight chain andbranched), and organic proton acceptors, (such as, for example,pyridine, triethylamine, N-methylmorpholine, andN,N-dimethylaminopyridine), and mixtures thereof. In some embodiments,the proton acceptor may be stabilized by a suitable counterion such aslithium, potassium, sodium, calcium, magnesium, and the like. In aspecific embodiment, the proton acceptor is triethylamine. The amount ofproton acceptor included in the reaction can and will vary, but can bereadily determined by a person of ordinary skill in the art.

Further, peptide coupling agents may also be added to the reaction. Apeptide coupling agent may be used as a carboxyl activating agent forthe coupling of primary amines to yield amide bonds. Non-limitingexamples of suitable peptide coupling agents include EDCI(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)), HOBt(Hydroxybenzotriazole), DCC (N,N′-Dicyclohexylcarbodiimide), HATU((1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate)), HBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumHexafluorophosphate), and TBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate).In an exemplary embodiment, EDCI(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)) and HOBt(Hydroxybenzotriazole) are added to the reaction.

In general, the volume to mass ratio of the solvent to melampomagnolicacid or amine derivative of MMB ranges from about 1:1 to about 100:1. Invarious embodiments, the volume to mass ratio of the solvent tomelampomagnolic acid or amine derivative of MMB may range from about 1:1to about 5:1, from about 5:1 to about 10:1, from about 10:1 to about20:1, from about 20:1 to about 30:1, from about 30:1 to about 40:1, fromabout 40:1 to about 50:1, from about 50:1 to about 60:1, from about 60:1to about 70:1, from about 70:1 to about 80:1, from about 80:1 to about90:1, or from about 90:1 to about 100:1. In exemplary embodiments, thevolume to mass ratio of the solvent to melampomagnolic acid or aminederivative of MMB may range from about 20:1 to about 30:1. In otherexemplary embodiments, the volume to mass ratio of the solvent tomelampomagnolic acid or amine derivative of MMB may range from about10:1 to about 20:1.

(ii) Step (a)—Reaction Conditions

In general, the reaction is conducted at a temperature that ranges fromabout 0° C. to about 50° C. In various embodiments, the reaction may beconducted at a temperature from about 0° C. to about 10° C., from about10° C. to about 20° C., from about 20° C. to about 30° C., from about30° C. to about 40° C., or from about 40° C. to about 50° C. In certainembodiments, the reaction may be conducted at a temperature of about 25°C. The reaction generally is conducted in an inert atmosphere (e.g.,under nitrogen or argon) and under ambient pressure.

Typically, the reaction is allowed to proceed for a sufficient period oftime until the reaction is complete, as determined by chromatography(e.g., TLC, HPLC) or another suitable method. In this context, a“completed reaction” generally means that the reaction mixture containsa significantly diminished amount of melampomagnolic acid, and asignificantly increased amount of the compound comprising Formula (I) or(VII) compared to the amounts of each present at the beginning of thereaction. Typically, the amount of melampomagnolic acid remaining in thereaction mixture after the reaction is complete may be less than about3%, or less than about 1%. In general, the reaction may proceed forabout 5 minutes to about 48 hours. Typically, the duration of thereaction is longer at lower reaction temperatures. In certainembodiments, the reaction may be allowed to proceed for about a periodof time ranging from about 5 minutes to about 10 minutes, from about 10minutes to about 15 minutes, from about 15 minutes to about 30 minutes,from about 30 minutes to about 1 hour, about 1 hour to about 3 hours,from about 3 hours to about 6 hours, from about 6 hours to about 12hours, from about 12 hours to about 18 hours, from about 18 hours toabout 24 hours, from about 24 hours to about 36 hours, or from about 36hours to about 48 hours. In certain embodiments, the reaction may beallowed to proceed about 3 hours to about 15 hours. In otherembodiments, the reaction may be allowed to proceed about 3 hours toabout 8 hours.

The yield of the compound comprising Formula (I), (VII) or (VIII) canand will vary. Typically, the yield of the compound comprising Formula(I), (VII) or (VIII) may be at least about 40%. In one embodiment, theyield of the compound comprising Formula (I), (VII) or (VIII) may rangefrom about 40% to about 60%. In another embodiment, the yield of thecompound comprising Formula (I), (VII) or (VIII) may range from about60% to about 80%. In a further embodiment, the yield of the compoundcomprising Formula (I), (VII) or (VIII) may range from about 80% toabout 90%. In still another embodiment, the yield of the compoundcomprising Formula (I), (VII) or (VIII) may be greater than about 90%,or greater than about 95%.

III. Compositions

The present disclosure also provides pharmaceutical compositions. Thepharmaceutical composition comprises a compound comprising Formula (I),(II), (III), (IV), (V), (VI), (VII) or (VIII) which is detailed above inSection I, as an active ingredient and at least one pharmaceuticallyacceptable excipient.

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pectin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isomorphous silicate, and microcrystalline cellulose.

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate,citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally, parenterally, or topically indosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, or intrasternal injection, or infusion techniques.Formulation of drugs is discussed in, for example, Gennaro, A. R.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(18^(th) ed, 1995), and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, and intraperitoneal), the preparation may bean aqueous or an oil-based solution. Aqueous solutions may include asterile diluent such as water, saline solution, a pharmaceuticallyacceptable polyol such as glycerol, propylene glycol, or other syntheticsolvents; an antibacterial and/or antifungal agent such as benzylalcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and thelike; an antioxidant such as ascorbic acid or sodium bisulfite; achelating agent such as etheylenediaminetetraacetic acid; a buffer suchas acetate, citrate, or phosphate; and/or an agent for the adjustment oftonicity such as sodium chloride, dextrose, or a polyalcohol such asmannitol or sorbitol. The pH of the aqueous solution may be adjustedwith acids or bases such as hydrochloric acid or sodium hydroxide.Oil-based solutions or suspensions may further comprise sesame, peanut,olive oil, or mineral oil.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Transmucosal administration may beaccomplished through the use of nasal sprays, aerosol sprays, tablets,or suppositories, and transdermal administration may be via ointments,salves, gels, patches, or creams as generally known in the art.

In certain embodiments, a composition comprising a compound of theinvention is encapsulated in a suitable vehicle to either aid in thedelivery of the compound to target cells, to increase the stability ofthe composition, or to minimize potential toxicity of the composition.As will be appreciated by a skilled artisan, a variety of vehicles aresuitable for delivering a composition of the present invention.Non-limiting examples of suitable structured fluid delivery systems mayinclude nanoparticles, liposomes, microemulsions, micelles, dendrimersand other phospholipid-containing systems. Methods of incorporatingcompositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery of the compound of the invention in view of their structuraland chemical properties. Generally speaking, liposomes are sphericalvesicles with a phospholipid bilayer membrane. The lipid bilayer of aliposome may fuse with other bilayers (e.g., the cell membrane), thusdelivering the contents of the liposome to cells. In this manner, thecompound of the invention may be selectively delivered to a cell byencapsulation in a liposome that fuses with the targeted cell'smembrane.

Liposomes may be comprised of a variety of different types ofphosolipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tetradecanoate (myristate),n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecadienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide,1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally, contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying the compound of the invention (i.e., having at leastone methionine compound) may be prepared by any known method ofpreparing liposomes for drug delivery, such as, for example, detailed inU.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561, 4,755,388, 4,828,837,4,925,661, 4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211 and5,264,618, the disclosures of which are hereby incorporated by referencein their entirety. For example, liposomes may be prepared by sonicatinglipids in an aqueous solution, solvent injection, lipid hydration,reverse evaporation, or freeze drying by repeated freezing and thawing.In a preferred embodiment the liposomes are formed by sonication. Theliposomes may be multilamellar, which have many layers like an onion, orunilamellar. The liposomes may be large or small. Continued high-shearsonication tends to form smaller unilamellar liposomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of methioninecompound, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. The composition ofthe invention may be encapsulated in a microemulsion by any methodgenerally known in the art.

In yet another embodiment, a composition of the invention may bedelivered in a dendritic macromolecule, or a dendrimer. Generallyspeaking, a dendrimer is a branched tree-like molecule, in which eachbranch is an interlinked chain of molecules that divides into two newbranches (molecules) after a certain length. This branching continuesuntil the branches (molecules) become so densely packed that the canopyforms a globe. Generally, the properties of dendrimers are determined bythe functional groups at their surface. For example, hydrophilic endgroups, such as carboxyl groups, would typically make a water-solubledendrimer. Alternatively, phospholipids may be incorporated in thesurface of a dendrimer to facilitate absorption across the skin. Any ofthe phospholipids detailed for use in liposome embodiments are suitablefor use in dendrimer embodiments. Any method generally known in the artmay be utilized to make dendrimers and to encapsulate compositions ofthe invention therein. For example, dendrimers may be produced by aniterative sequence of reaction steps, in which each additional iterationleads to a higher order dendrimer. Consequently, they have a regular,highly branched 3D structure, with nearly uniform size and shape.Furthermore, the final size of a dendrimer is typically controlled bythe number of iterative steps used during synthesis. A variety ofdendrimer sizes are suitable for use in the invention. Generally, thesize of dendrimers may range from about 1 nm to about 100 nm.

IV. Methods for Inhibiting Cancer Cell Growth

A further aspect of the present disclosure provides a method forinhibiting growth of a cancer cell. Cancer cell growth includes cellproliferation and cell metastasis. The method comprises contacting thecancer cell with an effective amount of a compound comprising Formula(I), (II), (III), (IV), (V), (VI), (VII) or (VIII), or apharmaceutically acceptable salt thereof, wherein the amount iseffective to inhibit growth of the cancer cell. Compounds comprisingFormula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII) are detailedabove in Section I. In some embodiments, the compound comprising Formula(I), (II), (III), (IV), (V), (VI), (VII) or (VIII) is used as part of acomposition, examples of which are detailed above in Section III.

(a) Contacting the Cell

In some embodiments, the cancer cell may be in vitro. The cancer cellmay be an established, commercially-available cancer cell line (e.g.,American Type Culture Collection (ATCC), Manassas, Va.). The cancer cellline may be derived from a blood cancer or a solid tumor. The cancercell line may be a human cell line or a mammalian cell line. In aspecific embodiment, the cancer cell line may be derived from a bloodcancer. In one exemplary embodiment, the cancer cell line may be derivedfrom a leukemic cell. The leukemic cell may be an acute myeloid leukemiacell, a chronic myeloid leukemia cell, an acute lymphocytic leukemiacell, a chronic lymphocytic leukemia cell, a cutaneous T cell leukemia,or another type of leukemia cell. In some embodiments, the cancer cellline may be a leukemia cell line such as CCRF-CEM, HL-60(TB), K-562,MOLT-4, RPMI-8226, or SR. In a specific embodiment, the cancer cell linemay be the leukemia cell line M9 ENL. In other embodiments, the cancercell line may be a hematopoietic or lymphoid cell line. Non-limitingexamples of hematopoietic or lymphoid cell lines include 380, 697,A3-KAW, A3/KAW, A4-Fuk, A4/Fuk, ALL-PO, ALL-SIL, AML-193, AMO-1, ARH-77,ATN-1, BALL-1, BC-3, BCP-1, BDCM, BE-13, BL-41, BL-70, BV-173, C8166,CA46, CCRF-CEM, CI-1, CMK, CMK-11-5, CMK-86, CML-T1, COLO 775, COLO-677,CTB-1, CTV-1, Daudi, DB, DEL, DG-75, DND-41, DOHH-2, EB1, EB2, EHEB,EJM, EM-2, EOL-1, EoL-1-cell, F-36P, GA-10, GA-10-Clone-4, GDM-1, GR-ST,GRANTA-519, H9, HAL-01, HD-MY-Z, HDLM-2, HEL, HEL 92.1.7, HH, HL-60,HPB-ALL, Hs 604.T, Hs 611.T, Hs 616.T, Hs 751.T, HT, HTK-, HuNS1, HuT102, HuT 78, IM-9, J-RT3-T3-5, JeKo-1, JiyoyeP-2003, JJN-3, JK-1, JM1,JURKAT, JURL-MK1, JVM-2, JVM-3, K-562, K052, KARPAS-299, KARPAS-422,KARPAS-45, KARPAS-620, KASUMI-1, KASUMI-2, Kasumi-6, KCL-22, KE-37,KE-97, KG-1, KHM-1B, Ki-JK, KM-H2, KMM-1, KMOE-2, KMS-11, KMS-12-BM,KMS-12-PE, KMS-18, KMS-20, KMS-21BM, KMS-26, KMS-27, KMS-28BM, KMS-34,KO52, KOPN-8, KU812, KY821, KYO-1, L-1236, L-363, L-428, L-540, LAMA-84,LC4-1, Loucy, LOUCY, LP-1, M-07e, MC-CAR, MC116, ME-1, MEC-1, MEC-2,MEG-01, MHH-CALL-2, MHH-CALL-3, MHH-CALL-4, MHH-PREB-1, Mino, MJ, ML-2,MLMA, MM1-S, MN-60, MOLM-13, MOLM-16, MOLM-6, MOLP-2, MOLP-8, MOLT-13,MOLT-16, MOLT-4, MONO-MAC-1, MONO-MAC-6, MOTN-1, MUTZ-1, MUTZ-3, MUTZ-5,MV-4-11, NALM-1, NALM-19, NALM-6, NAMALWA, NB-4, NCI-H929, NCO2, NKM-1,NOMO-1, NU-DHL-1, NU-DUL-1, OCI-AML2, OCI-AML3, OCI-AML5, OCI-LY-19,OCI-LY10, OCI-LY3, OCI-M1, OPM-2, P12-ICHIKAWA, P30-OHK, P31-FUJ,P31/FUJ, P3HR-1, PCM6, PEER, PF-382, Pfeiffer, PL-21, Raji,Ramos-2G6-4C10, RCH-ACV, REC-1, Reh, REH, RI-1, RL, RPMI 8226,RPMI-8226, RPMI-8402, RS4-11, “RS4;11”, SEM, Set-2, SIG-M5, SK-MM-2,SKM-1, SR, SR-786, ST486, SU-DHL-1, SU-DHL-10, SU-DHL-4, SU-DHL-5,SU-DHL-6, SU-DHL-8, SUP-B15, SUP-B8, SUP-HD1, SUP-M2, SUP-T1, SUP-T11,TALL-1, TF-1, THP-1, TO 175.T, Toledo, TUR, U-266, U-698-M, U-937,U266B1, UT-7, WSU-DLCL2, and WSU-NHL.

In another exemplary embodiment, the cancer cell line may be derivedfrom a solid tumor cell. The solid tumor cell may be a non-small celllung cancer, colon cancer, CNS cancer, melanoma cancer, ovarian cancer,renal cancer, prostate cancer, breast cancer, or another type of solidtumor cell. In some embodiments, the cancer cell line may be a non-smallcell lung cancer cell line such as A549/ATCC, HOP-92, NCI-H226, NCI-H23,NCI-H322M, NCI-H460, or NCI-H522. In other embodiments, the cancer cellline may be a colon cancer cell line such as COLO 205, HCC-2998,HCT-116, HCT-15, HT29, KM12 or SW-620. In different embodiments, thecancer cell line may be a CNS cancer cell line such as SF-268, SF-295,SF-539, SNB-19, SNB-75 or U251. In some other embodiments, the cancercell line may be a melanoma cell line such as LOX IMVI, MALME-3M, M14,MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, or UACC-62. Instill other embodiments, the cancer cell line may be an ovarian cancercell line such as OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES,IGROV1 or SK-OV-3. In some different embodiments, the cancer cell linemay be a renal cancer cell line such as 786-0, A498, ACHN, CAKI-1, RXF393, SN12C, TK-10, or UO-31. In other embodiments, the cancer cell linemay be a prostate cancer cell line such as PC-3 or DU-145. In someembodiments, the cancer cell line may be a breast cancer cell line suchas MCF7, MDA-BM-231/ATCC, HS 578T, BT-549, T-47D, or MDA-MB-468.

In other embodiments, the cancer cell may be in vivo; i.e., the cell maybe disposed in a subject. In such embodiments, the cancer cell iscontacted with the compound comprising Formula (I), (II), (III), (IV),(V), (VI), (VII) or (VIII) by administering the compound comprisingFormula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII) to thesubject. In some embodiments, the subject may be a human. In otherembodiments, the subject may be a non-human animal. Non-limitingexamples of non-human animals include companion animals (e.g., cats,dogs, horses, rabbits, gerbils), agricultural animals (e.g., cows, pigs,sheep, goats, fowl), research animals (e.g., rats, mice, rabbits,primates), and zoo animals (e.g., lions, tiger, elephants, and thelike).

The cancer cell disposed in the subject may be a blood cancer cell(e.g., leukemia, lymphoma, myeloma) or a solid tumor cancer cell. Thecancer may be primary or metastatic; early stage or late stage; and/orthe tumor may be malignant or benign. Non-limiting cancers includebladder cancer, bone cancer, brain cancer, breast cancer, centralnervous system cancer, cervical cancer, colon cancer, colorectal cancer,duodenal cancer, endometrial cancer, esophageal cancer, eye cancer,gallbladder cancer, germ cell cancer, kidney cancer, larynx cancer,leukemia, liver cancer, lymphoma, lung cancer, melanoma, mouth/throatcancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer,testicular cancer, thyroid cancer, vaginal cancer, and drug resistantcancers. In one exemplary embodiment, the cancer cell may be a leukemia.The leukemia may be an acute lymphocytic (lymphoblastic) leukemia, achronic lymphocytic leukemia, an acute myeloid leukemia, a chronicmyeloid leukemia, a hairy cell leukemia, a T-cell prolymphocyticleukemia, a large granular lymphocytic leukemia, or an adult T-cellleukemia. In another exemplary embodiment, the cancer cell may be asolid tumor cancer cell selected from the group consisting of non-smallcell lung cancer, colon cancer, CNS cancer, melanoma cancer, ovariancancer, renal cancer, prostate cancer and breast cancer.

The compound comprising Formula (I), (II), (III), (IV), (V), (VI), (VII)or (VIII) may be administered to the subject orally (as a solid or aliquid), parenterally (which includes intramuscular, intravenous,intradermal, intraperitoneal, and subcutaneous), or topically (whichincludes transmucosal and transdermal). An effective amount of thecompound can be determined by a skilled practitioner in view of desireddosages and potential side effects of the compound.

The compound comprising Formula (I), (II), (III), (IV), (V), (VI), (VII)or (VIII) may be administered once or administered repeatedly to thesubject. Repeated administrations may be at regular intervals of 2hours, 6 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 30 days, andso forth.

(b) Inhibiting Cancer Cell Growth

Following contact with an effective amount of the compound comprisingFormula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII) growth of thecancer cell is inhibited. Cell growth or proliferation can be measuredin cells grown in vitro using standard cell viability or cellcytotoxicity assays (e.g., based on DNA content, cell permeability,etc.) in combination with cell counting methods (e.g., flow cytometry,optical density). Cell growth or proliferation can be measured in vivousing imaging procedures and/or molecular diagnostic indicators.

In an embodiment, contact with an effective amount of the compoundcomprising Formula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII)selectively inhibits growth of cancer cells. As such, a compoundcomprising Formula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII)does not appreciably kill non-cancer cells at the same concentration.Accordingly, more than 50% of non-cancer cells remain viable followingcontact with a compound comprising Formula (I), (II), (III), (IV), (V),(VI), (VII) or (VIII) at the same concentration. For example about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95% or about 100% of non-cancer cells remainviable following contact with a compound comprising Formula (I), (II),(III), (IV), (V), (VI), (VII) or (VIII) at the same concentration. Or,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of non-cancercells remain viable following contact with a compound comprising Formula(I), (II), (III), (IV), (V), (VI), (VII) or (VIII) at the sameconcentration.

In various embodiments, cancer cell growth may be inhibited about0.5-fold, about 1-fold, about 2-fold, about 3-fold, about 4-fold, about5-fold, about 8-fold, about 10-fold, or more than 10-fold relative to areference value. In various other embodiments, cancer cell growth may beinhibited 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold,10-fold, or more than 10-fold relative to a reference value. In otherembodiments, cancer cell growth may be inhibited to such a degree thatthe cell undergoes cell death (via apoptosis or necrosis). Any suitablereference value known in the art may be used. For example, a suitablereference value may be cancer cell growth in a sample that has not beencontacted with a compound comprising Formula (I), (II), (III), (IV),(V), (VI), (VII) or (VIII). In another example, a suitable referencevalue may be the baseline growth rate of the cells as determined bymethods known in the art. In another example, a suitable reference valuemay be a measurement of the number of cancer cells in a reference sampleobtained from the same subject. For example, when monitoring theeffectiveness of a therapy or efficacy of a compound comprising Formula(I), (II), (III), (IV), (V), (VI), (VII) or (VIII), a reference samplemay be a sample obtained from a subject before therapy or administrationof the compound comprising Formula (I), (II), (III), (IV), (V), (VI),(VII) or (VIII) began.

(c) Optional Contact

In certain embodiments, the method may further comprise contacting thecell with at least one chemotherapeutic agent and/or a radiotherapeuticagent. The chemotherapeutic agent and/or radiotherapeutic agent may beadministered concurrently or sequentially with the compound comprisingFormula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII).

The chemotherapeutic agent may be an alkylating agent, ananti-metabolite, an anti-tumor antibiotic, an anti-cytoskeletal agent, atopoisomerase inhibitor, an anti-hormonal agent, a targeted therapeuticagent, or a combination thereof. Non-limiting examples of suitablealkylating agents include altretamine, benzodopa, busulfan, carboplatin,carboquone, carmustine (BCNU), chlorambucil, chlornaphazine,cholophosphamide, chlorozotocin, cisplatin, cyclosphosphamide,dacarbazine (DTIC), estramustine, fotemustine, ifosfamide, improsulfan,lomustine (CCNU), mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, meturedopa, nimustine, novembichin, phenesterine, piposulfan,prednimustine, ranimustine; temozolomide, thiotepa, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphaoramide,trimethylolomelamine, trofosfamide, uracil mustard and uredopa. Suitableanti-metabolites include, but are not limited to aminopterin,ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur(1-hexylcarbomoyl-5-fluorouracil), cladribine, cytarabine or cytosinearabinoside (Ara-C), dideoxyuridine, denopterin, doxifluridine,enocitabine, floxuridine, fludarabine, 5-fluorouracil, gemcetabine,hydroxyurea, leucovorin (folinic acid), 6-mercaptopurine, methotrexate,pemetrexed, pteropterin, thiamiprine, trimetrexate, and thioguanine.Non-limiting examples of suitable anti-tumor antibiotics includeaclacinomysin, actinomycins, adriamycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mithramycin, mycophenolic acid,nogalamycin, olivomycins, peplomycin, plicamycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, valrubicin, ubenimex, zinostatin, and zorubicin.Non-limiting examples of suitable anti-cytoskeletal agents includecolchicines, docetaxel, macromycin, paclitaxel, vinblastine,vincristine, vindesine, and vinorelbine. Suitable topoisomeraseinhibitors include, but are not limited to, amsacrine, etoposide(VP-16), irinotecan, mitoxantrone, RFS 2000, teniposide, and topotecan.Non-limiting examples of suitable anti-hormonal agents such asaminoglutethimide, aromatase inhibiting 4(5)-imidazoles, bicalutamide,finasteride, flutamide, goserelin, 4-hydroxytamoxifen, keoxifene,leuprolide, LY117018, mitotane, nilutamide, onapristone, raloxifene,tamoxifen, toremifene, and trilostane. Examples of targeted therapeuticagents include, without limit, monoclonal antibodies such asalemtuzumab, epratuzumab, gemtuzumab, ibritumomab tiuxetan, rituximab,tositumomab, and trastuzumab; protein kinase inhibitors such asbevacizumab, cetuximab, dasatinib, erlotinib, gefitinib, imatinib,lapatinib, mubritinib, nilotinib, panitumumab, pazopanib, sorafenib,sunitinib, and vandetanib; angiogeneisis inhibitors such as angiostatin,endostatin, bevacizumab, genistein, interferon alpha, interleukin-2,interleukin-12, pazopanib, pegaptanib, ranibizumab, rapamycin,thalidomide; and growth inhibitory polypeptides such as erythropoietin,interleukins (e.g., IL-1, IL-2, IL-3, IL-6), leukemia inhibitory factor,interferons, thrombopoietin, TNF-α, CD30 ligand, 4-1 BB ligand, andApo-1 ligand. Also included are pharmaceutically acceptable salts,acids, or derivatives of any of the above listed agents. The mode ofadministration of the chemotherapeutic agent can and will vary dependingupon the agent and the type of cancer. A skilled practitioner will beable to determine the appropriate dose of the chemotherapeutic agent.

The radiotherapeutic agent may include a radioisotope. Suitableradioisotopes include, without limit, Iodine-131, Iodine-125,Iodine-124, Lutecium-177, Phosphorous-132, Rhenium-186, Strontium-89,Yttrium-90, Iridium-192, and Samarium-153. Alternatively, theradiotherapeutic agent may include a high Z-element chosen from gold,silver, platinum, palladium, cobalt, iron, copper, tin, tantalum,vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium,thallium, lead, bismuth, gadolinium, dysprosium, holmium, and uranium.The appropriate dose of the radiotherapeutic agent may be determined bya skilled practitioner.

Definitions

The compounds described herein have asymmetric centers. Compounds of thepresent invention containing an asymmetrically substituted atom may beisolated in optically active or racemic form. All chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated.

The term “acyl,” as used herein alone or as part of another group,denotes the moiety formed by removal of the hydroxy group from the groupCOOH of an organic carboxylic acid, e.g., RC(O)—, wherein R is R¹, R¹O—,R¹R²N, or R¹S—, R¹ is hydrocarbyl, heterosubstituted hydrocarbyl, orheterocyclo, and R² is hydrogen, hydrocarbyl, or substitutedhydrocarbyl.

The term “acyloxy,” as used herein alone or as part of another group,denotes an acyl group as described above bonded through an oxygenlinkage (O), e.g., RC(O)O— wherein R is as defined in connection withthe term “acyl.”

The term “aliphatic” as used herein alone or as part of another groupdenotes an optionally substituted straight chain, branched chain ornon-aromatic ring (alicyclic). These aliphatic compounds may besaturated (alkanes) or unsaturated (alkenes or alkynes). Besideshydrogen, other elements can be bound to the carbon chain. By way ofnon-limiting example: oxygen, nitrogen, sulfur and chlorine. Nonlimiting examples of an aliphatic group may be methane, ethyne, ethane,ethane, propyne, propene, propane, 1,2-butadiene, 1-butyne, butane,butane, cyclochexene, n-pentane, cycloheptane, methylcyclohexane,cubane, nonane, dicyclopentadiene, phellandrene, α-terpinene, limonene,undecane, squalene and polyethylene.

The term “alkyl” as used herein describes groups which are preferablylower alkyl containing from one to eight carbon atoms in the principalchain and up to 20 carbon atoms. They may be straight or branched chainor cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl andthe like.

The term “alkenyl” as used herein describes groups which are preferablylower alkenyl containing from two to eight carbon atoms in the principalchain and up to 20 carbon atoms. They may be straight or branched chainor cyclic and include ethenyl, propenyl, isopropenyl, butenyl,isobutenyl, hexenyl, and the like.

The term “alkynyl” as used herein describes groups which are preferablylower alkynyl containing from two to eight carbon atoms in the principalchain and up to 20 carbon atoms. They may be straight or branched chainand include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and thelike.

The term “amine” as used herein describes a primary, secondary, tertiaryor cyclic amine. An amine may be an alkylamine, an arylamine, analkylarylamine, an aliphatic amine or an aromatic amine. A primary aminehas one of three hydrogen atoms replaced by an alkyl or aromatic.Non-limiting examples of primary alkyl amines include methylamine,ethanolamine (2-aminoethanol), and tris, while primary aromatic aminesinclude aniline. A secondary amines has two organic substituents (alkyl,aryl or both) bound to N together with one hydrogen (or no hydrogen ifone of the substituent bonds is double). Non-limiting examples includedimethylamine and methylethanolamine, while an example of an aromaticamine would be diphenylamine. A tertiary amines has all three hydrogenatoms replaced by organic substituents. Examples include trimethylamineor triphenylamine. A cyclic amine is either a secondary or a tertiaryamine. Examples of cyclic amines include the 3-member ring aziridine andthe six-membered ring piperidine. N-methylpiperidine andN-phenylpiperidine are examples of cyclic tertiary amines.

The term “aromatic” as used herein alone or as part of another groupdenotes optionally substituted homo- or heterocyclic conjugated planarring or ring system comprising delocalized electrons. These aromaticgroups are preferably monocyclic (e.g., furan or benzene), bicyclic, ortricyclic groups containing from 5 to 14 atoms in the ring portion. Theterm “aromatic” encompasses “aryl” groups defined below.

The terms “aryl” or “Ar” as used herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 10 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl, or substituted naphthyl.

The terms “carbocyclo” or “carbocyclic” as used herein alone or as partof another group denote optionally substituted, aromatic ornon-aromatic, homocyclic ring or ring system in which all of the atomsin the ring are carbon, with preferably 5 or 6 carbon atoms in eachring. Exemplary substituents include one or more of the followinggroups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl,acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal,carbamyl, carbocyclo, cyano, ester, ether, halogen, heterocyclo,hydroxy, keto, ketal, phospho, nitro, and thio.

The terms “halogen” or “halo” as used herein alone or as part of anothergroup refer to chlorine, bromine, fluorine, and iodine.

The term “heteroatom” refers to atoms other than carbon and hydrogen.

The term “heteroaromatic” as used herein alone or as part of anothergroup denotes optionally substituted aromatic groups having at least oneheteroatom in at least one ring, and preferably 5 or 6 atoms in eachring. The heteroaromatic group preferably has 1 or 2 oxygen atoms and/or1 to 4 nitrogen atoms in the ring, and is bonded to the remainder of themolecule through a carbon. Exemplary groups include furyl, benzofuryl,oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzoxadiazolyl,pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl,pyrimidyl, pyrazinyl, pyridazinyl, indolyl, isoindolyl, indolizinyl,benzimidazolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl,carbazolyl, purinyl, quinolinyl, isoquinolinyl, imidazopyridyl, and thelike. Exemplary substituents include one or more of the followinggroups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl,acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal,carbamyl, carbocyclo, cyano, ester, ether, halogen, heterocyclo,hydroxy, keto, ketal, phospho, nitro, and thio.

The terms “heterocyclo” or “heterocyclic” as used herein alone or aspart of another group denote optionally substituted, fully saturated orunsaturated, monocyclic or bicyclic, aromatic or non-aromatic groupshaving at least one heteroatom in at least one ring, and preferably 5 or6 atoms in each ring. The heterocyclo group preferably has 1 or 2 oxygenatoms and/or 1 to 4 nitrogen atoms in the ring, and is bonded to theremainder of the molecule through a carbon or heteroatom. Exemplaryheterocyclo groups include heteroaromatics as described above. Exemplarysubstituents include one or more of the following groups: hydrocarbyl,substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy, alkenyl,alkenoxy, aryl, aryloxy, amino, amido, acetal, carbamyl, carbocyclo,cyano, ester, ether, halogen, heterocyclo, hydroxy, keto, ketal,phospho, nitro, and thio.

The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms.

The term “indole” as used herein denotes an aromatic heterocyclicorganic compound. The indole has a bicyclic structure, consisting of asix-membered benzene ring fused to a five-membered nitrogen-containingpyrrole ring. The indole may be optionally substituted at any positionalong the benzene ring. Additionally, the indole may be substituted atany position along the pyrrole ring. Further, the nitrogen of thepyrrole may be substituted with another element. Non-limiting examplesmay include sulfur or oxygen.

The term “oxygen-protecting group” as used herein denotes a groupcapable of protecting an oxygen atom (and hence, forming a protectedhydroxyl group), wherein the protecting group may be removed, subsequentto the reaction for which protection is employed, without disturbing theremainder of the molecule. Exemplary oxygen protecting groups includeethers (e.g., allyl, triphenylmethyl (trityl or Tr), p-methoxybenzyl(PMB), p-methoxyphenyl (PMP)); acetals (e.g., methoxymethyl (MOM),β-methoxyethoxymethyl (MEM), tetrahydropyranyl (THP), ethoxy ethyl (EE),methylthiomethyl (MTM), 2-methoxy-2-propyl (MOP),2-trimethylsilylethoxymethyl (SEM)); esters (e.g., benzoate (Bz), allylcarbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethylcarbonate); silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl(TES), triisopropylsilyl (TIPS), triphenylsilyl (TPS),t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS)) and thelike. A variety of oxygen protecting groups and the synthesis thereofmay be found in “Protective Groups in Organic Synthesis” by T. W. Greeneand P. G. M. Wuts, 3^(rd) ed., John Wiley & Sons, 1999.

The “substituted hydrocarbyl” moieties described herein are hydrocarbylmoieties which are substituted with at least one atom other than carbon,including moieties in which a carbon chain atom is substituted with aheteroatom such as nitrogen, oxygen, silicon, phosphorous, boron, or ahalogen atom, and moieties in which the carbon chain comprisesadditional substituents. These substituents include alkyl, alkoxy, acyl,acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal,carbamyl, carbocyclo, cyano, ester, ether, halogen, heterocyclo,hydroxy, keto, ketal, phospho, nitro, and thio.

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples represent techniquesdiscovered by the inventors to function well in the practice of theinvention. Those of skill in the art should, however, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth is to be interpreted asillustrative and not in a limiting sense.

Example 1. JVM 1

To a stirred solution of MMB (50 mg, 0.189 mmol) in dichloromethane, wasadded carbonyldiimidazole (46.02 mg, 0.284 mmol). The reaction mixturewas stirred at ambient temperature for 24 hours. After completion of thereaction, the reaction mixture was diluted with chloroform (2 mL). Theorganic layer was washed with 10% citric acid solution (2 mL), driedover Na₂SO₄, and concentrated under reduced pressure. The residue waspurified by column chromatography (silica gel eluted with 3% methanol indichloromethane) to afford compound JVM 1 as an off-white solid (yield:75%).

¹H-NMR (CDCl₃, 400 MHz) δ 8.13 (s, 1H), 7.68 (s, 1H), 7.42 (s, 1H),7.09-7.05 (m, 3H), 5.72 (t, J=8 Hz, 1H), 4.94 (d, J=12 Hz, 1H), 4.55 (d,J=12.8 Hz, 2H), 4.36 (d, J=16 Hz, 1H), 3.902-3.856 (m, 1H), 2.79 (t,J=11.8 Hz, 1H), 2.60 (d, J=8 Hz, M), 2.44-1.91 (m, 8H), 1.83-1.74 (m,1H), 1.66 (t, J=12 Hz, 1H), 1.52 (s, 1H), 1.07 (t, J=12 Hz, 1H); ¹³C NMR(CDCl₃, 100 MHz) δ 174.5, 148.7, 137.7, 137.0, 133.3, 132.7, 130.9,130.6, 119.4, 117.1, 80.8, 69.8, 62.4, 59.9, 48.3, 43.7, 40.9, 36.2,26.5, 23.8, 23.6, 17.8 ppm.

Example 2. JVM 57 (6d in Example 19)

4-(2-Aminoethyl)morpholine (25 mg, 0.19 mmol) in dichloromethane (2 mL)was added at 0° C. to the triazole intermediate of MMB (JVM 2-16) (70mg, 0.19 mmol). The reaction mixture was stirred for 15 hours. Uponcompletion of the reaction, as determined by TLC, water was added to thereaction mixture and the resulting aqueous mixture was extracted withdichloromethane. The organic layer was washed with water followed bybrine, dried over anhydrous Na₂SO₄, and concentrated to afford the crudeproduct. The crude product was purified by column chromatography (silicagel eluted with 3% methanol in dichloromethane) to afford compound JVM57 as a white solid (yield: 60%).

¹H NMR (CDCl₃, 400 MHz): δ 6.11 (d, J=3.6 Hz, 1H), 5.58 (t, J=8 Hz, 1H),5.43 (d, J=2.8 Hz, 1H), 5.06 (s, 1H), 4.53 (d, J=12.4 Hz, 1H), 4.34 (d,J=12 Hz, 1H), 3.74 (t, J=8.8 Hz, 1H), 3.56 (s, 4H), 3.16 (t, J=5.2 Hz,2H), 2.78-2.72 (m, 2H), 2.32 (s, 8H), 2.21-2.00 (m, 4H), 1.55 (t, J=10.4Hz, 1H), 1.41 (s, 3H), 1.01 (t, J=12 Hz, 1H) ppm. ¹³C NMR (CDCl3, 100MHz) δ 169.2, 155.9, 138.7, 135.4, 130.2, 120.0, 80.9, 67.0, 66.7, 63.1,59.8, 57.2, 53.1, 42.5, 37.0, 36.5, 25.7, 24.4, 23.7, 17.8 ppm.

Example 3. JVM 59 (6b in Example 19)

To the triazole intermediate of MMB (JVM 2-16) (70 mg, 0.19 mmol) indichloromethane (2 mL) at 0° C. was added 1-(2-aminoethyl)pyrrolidine(21.6 mg, 0.19 mmol). The reaction mixture was stirred for 16 hours.Upon completion of the reaction, as determined by TLC, water was addedto the reaction mixture and the aqueous mixture was extracted withdichloromethane. The organic layer was washed with water followed bybrine, dried over anhydrous Na₂SO₄, and concentrated to afford the crudeproduct. The crude product was purified by column chromatography (silicagel eluted with 5% methanol in dichloromethane) to afford compound JVM59 as a white solid (yield: 55%).

¹H NMR (CDCl₃, 400 MHz): δ 6.22 (d, J=3.2 Hz, 1H), 5.65 (t, J=7 Hz, 1H),5.55 (s, 1H), 4.62 (d, J=11.6 Hz, 1H), 4.47 (d, J=12.4 Hz, 1H), 3.82 (t,J=9.6 Hz, 1H), 3.35 (s, 2H), 2.90-2.83 (m, 2H), 2.70 (s, 4H), 2.42 (d,J=9.6 Hz, 2H), 2.38-2.13 (m, 7H) 1.84 (s, 4H), 1.66 (t, J=12 Hz, 1H),1.52 (s, 3H), 1.13 (t, J=11.6 Hz, 1H) ppm. ¹³C NMR (CDCl3, 100 MHz) δ169.3, 156.1, 138.6, 135.3, 129.8, 120.1, 80.9, 67.0, 63.1, 59.8, 55.1,53.8, 42.5, 38.9, 36.5, 25.6, 24.3, 23.6, 23.2, 17.8 ppm.

Example 4. JVM 61 (6c in Example 19)

To the triazole intermediate of MMB (JVM 2-16) (70 mg, 0.19 mmol) indichloromethane (2 mL), 2-ethylaminopyridine (23.18 mg, 0.19 mmol) wasadded at 0° C. The reaction mixture was stirred for 16 hours. Uponcompletion as determined by TLC, water was added to the reaction mixtureand the aqueous mixture was extracted with dichloromethane. The organiclayer was washed with water followed by brine, dried over anhydrousNa₂SO₄, and concentrated to afford the crude product. The crude productwas purified by column chromatography (silica gel eluted with 5%methanol in dichloromethane) to afford compound JVM 61 as a white solid(yield: 65%).

¹H NMR (CDCl₃, 400 MHz): δ 8.50 (s, 1H), 7.62 (t, J=7.2 Hz, 1H), 7.15(d, J=7.6 Hz, 2H), 6.19 (s, 1H), 5.64 (t, J=8 Hz, 1H), 5.56 (m, 2H),4.62 (d, J=12.4 Hz, 1H), 4.44 (d, J=12.8 Hz, 1H), 3.84 (t, J=9.2 Hz,1H), 3.60 (d, J=6 Hz, 2H), 2.99-2.82 (m, 4H), 2.41-2.12 (m, 5H), 1.75(s, 1H), 1.64 (d, J=10.4 Hz, 1H), 1.52 (s, 3H), 1.07 (t, J=13.6 Hz, 1H).¹³C NMR (CDCl₃, 100 MHz): δ 169.3, 159.1, 156.0, 149.1, 138.6, 136.6,135.5, 129.9, 123.4, 121.6, 120.2, 81.0, 67.0, 63.2, 59.8, 42.5, 40.1,37.1, 36.6, 25.8, 24.4, 23.7, 17.9 ppm.

Example 5. JVM 64 (6e in Example 19)

To the triazole intermediate of MMB (JVM 2-16) (70 mg, 0.19 mmol) indichloromethane (2 mL), 5-(methylthio)-1H-1,2,4-triazol-3-amine (24.7mg, 0.19 mmol) was added at 0° C. The reaction mixture was stirred for 8hours. Upon completion as determined by TLC, water was added to thereaction mixture and the aqueous mixture was extracted withdichloromethane. The organic layer was washed with water followed bybrine, dried over anhydrous Na₂SO₄, and concentrated to afford the crudeproduct. The crude product was purified by column chromatography (silicagel eluted with 3% methanol in dichloromethane) to afford compound JVM64 as a white solid (yield: 62%).

¹H NMR (CDCl₃, 400 MHz): δ 6.22 (s, 2H), 5.82 (t, J=8 Hz, 1H), 5.50 (s,1H), 4.90 (d, J=12.4 Hz, 1H), 4.81 (d, J=12.4 Hz, 1H), 3.85 (t, 9.6 Hz,1H), 2.95 (s, 1H), 2.85 (d, J=9.2 Hz, 1H), 2.48-2.15 (m, 8H), 1.70-1.53(m, 6H), 1.13 (t, J=12.4 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ 169.1,163.1, 157.4, 149.9, 138.5, 133.3, 132.7, 120.2, 80.7, 70.1, 62.9, 59.7,42.4, 36.2, 25.6, 24.3, 23.7, 17.8, 13.5 ppm.

Example 6. JVM 66

To a stirred solution of MMB (50 mg, 0.19 mmol) in chloroform (2 mL),thiocarbonyldiimidazole (33.8 mg, 0.19 mmol) was added at ambienttemperature. The reaction was maintained for 1 hour at ambienttemperature. Upon completion as determined by TLC, water was added andthe aqueous mixture was extracted with dichloromethane. The organiclayer was dried over anhydrous Na₂SO₄ and concentrated to afford thecrude product. The crude product was purified by column chromatography(silica gel eluted with a gradient of 3-8% methanol in dichloromethane)to afford compound JVM 66 as an off-white solid (yield: 60%) andcompound JVM 66A as a white solid (yield: 15%).

¹H NMR (CDCl₃, 400 MHz): δ 8.11 (s, 1H), 7.38 (s, 1H), 7.07 (s, 1H),6.22 (s, 1H), 5.52 (s, 2H), 5.14 (s, 1H), 4.30 (s, 1H), 3.78 (t, J=8.8Hz, 1H), 3.16 (s, 1H), 2.94 (d, J=8.8 Hz, 1H), 2.47 (q, J=17.2 Hz, 2H),2.3-2.18 (m, 3H), 1.77-1.66 (m, 2H), 1.39 (s, 3H), 1.32-1.23 (m, 1H);¹³C NMR (CDCl₃, 100 MHz): δ 168.9, 165.1, 143.8, 138.7, 135.2, 131.0,119.8, 116.4, 115.6, 79.4, 63.0, 59.8, 53.3, 45.1, 37.4, 29.8, 28.2,24.7, 17.8 ppm.

Example 7. JVM 67

To a reaction mixture of MMB (200 mg, 0.76 mmol) and triethylamine (76.7mg, 0.76 mmol) in dichloromethane (5 mL), succinic anhydride (76 mg,0.76 mmol) was added at ambient temperature. The resulting reactionmixture was stirred for 48 hours. Upon completion as determined by TLC,the reaction mixture was concentrated under reduced pressure to affordthe crude product. The crude product was purified by columnchromatography (silica gel eluted with a gradient of 3-5% methanol indichloromethane) to afford compound JVM 67 as a white solid (yield:90%).

¹H NMR (DMSO-d₆, 400 MHz): δ 12.25 (s, 1H), 6.05 (d, J=2.8 Hz, 1H),5.64-5.57 (m, 2H), 4.64 (d, J=12.4 Hz, 1H), 4.42 (d, J=12.8 Hz, 1H),4.12 (t, J=9.6 Hz, 1H), 2.99 (t, J=3 Hz, 1H), 2.85 (d, J=9.6 Hz, 1H),2.30-2.04 (m, 10H), 1.66 (t, J=11.6 Hz, 1H), 1.47 (s, 3H), 0.96 (t,J=11.6 Hz, 1H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 173.8, 172.4, 169.8,140.0, 135.3, 129.5, 119.7, 110.0, 81.0, 66.9, 63.0, 60.3, 42.2, 36.7,29.1, 25.0, 24.2, 23.6, 17.9 ppm.

Example 8. JVM 88

To a stirred solution of MMB (50 mg, 0.19 mmol) in dichloromethane (2mL), was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 43.93mg, 0.23 mmol), triethylamine (48.4 mg, 0.48 mmol),dimethylaminopyridine (DMAP, 2.3 mg, 0.019 mmol) and1-piperidylpropionic acid (29.8 mg, 0.19 mmol) at 0° C. The reactionmixture was stirred at ambient temperature for 24 hours. Uponcompletion, water was added and the mixture was extracted withdichloromethane. The organic layer was washed with water followed bybrine, dried over anhydrous Na₂SO₄, and concentrated to afford the crudeproduct. The crude product was purified by column chromatography (silicagel eluted with 2% methanol in dichloromethane) to afford compound JVM88 as a white solid (yield: 62%).

¹H NMR (CDCl₃, 400 MHz): δ 6.23 (d, J=3.6 Hz, 1H), 5.65 (t, J=8 Hz, 1H),5.52 (d, J=3.2 Hz, 1H), 4.69 (d, J=12.4 Hz, 1H), 4.42 (d, J=12.4 Hz,1H), 3.84 (t, J=9.2 Hz, 1H), 2.85-2.81 (m, 2H), 2.65-2.62 (m, 2H),2.52-2.48 (m, 2H), 2.42-2.12 (m, 10H), 1.63-1.52 (m, 8H), 1.41 (d, J=4.4Hz, 2H), 1.10 (t, J=12 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ 172.4,169.4, 138.8, 135.0, 130.5, 120.4, 81.1, 66.6, 63.4, 60.0, 54.4, 54.3,42.7, 36.7, 32.3, 25.9, 25.8, 24.4, 24.2, 23.9, 18.1 ppm.

Example 9. JVM 96

To a reaction mixture of MMB carboxylic acid (JVM 67), (50 mg, 0.14mmol), EDC (40.26 mg, 0.21 mmol), N-hydroxybenzotriazole (HOBt, 28.35mg, 0.21 mmol), and triethylamine (42.42 mg, 0.42 mmol) indichloromethane (2 mL) was added 3-aminomethyl-6-chloropyridine (19.96mg, 0.14 mmol) at 0° C. and the reaction mixture was stirred at ambienttemperature for 16 hours. Upon completion as determined by TLC, waterwas added and the mixture extracted with dichloromethane. The organiclayer was dried and concentrated to afford the crude compound. The crudeproduct was further purified by column chromatography (silica gel using3% methanol in dichloromethane) to afford JVM 96 as pure product aswhite solid (yield 75%).

¹H NMR (CDCl₃, 400 MHz): δ 8.28 (s, 1H), 7.61 (d, J=8.4 Hz, 1H), 7.29(d, J=8.4 Hz, 1H), 6.22 (d, J=3.2 Hz, 1H), 6.07 (s, 1H), 5.68 (t, J=7.2Hz, 1H), 5.55 (s, 1H), 4.65 (d, J=12.4 Hz, 1H), 4.49 (d, J=12.8 Hz, 1H),4.42 (d, J=5.6 Hz, 2H), 3.85 (t, J=9.6 Hz, 1H), 2.95 (t, J=8.8 Hz, 1H),2.85 (d, J=9.6 Hz, 1H), 2.68 (t, J=6 Hz, 2H), 2.52-2.11 (m, 7H),1.67-1.62 (m, 2H), 1.53 (s, 3H), 1.12 (t, J=11.6 Hz, 1H); ¹³C NMR(CDCl₃, 100 MHz): δ 172.8, 171.5, 169.6, 150.7, 148.9, 138.9, 138.6,134.8, 133.1, 130.8, 124.4, 120.4, 81.2, 67.2, 63.3, 60.1, 42.7, 40.5,36.7, 30.7, 29.3, 25.8, 24.6, 23.9, 18.1 ppm.

Example 10. JVM 2-16

To a stirred solution of MMB (50 mg, 0.18 mmol) in dichloromethane,carbonylditriazole (46.5 mg, 0.28 mmol) was added at ambienttemperature. The reaction mixture was stirred at ambient temperature for10 minutes. Upon completion, water was added and the mixture extractedwith dichloromethane, dried over Na₂SO₄ and concentrated under reducedpressure to afford pure product JVM 2-16 as white solid (yield: 85%).

¹H NMR (CDCl₃, 400 MHz): δ 8.83 (s, 1H), 8.06 (s, 1H), 6.26 (d, J=3.6Hz, 1H), 5.92 (t, J=8.4 Hz, 1H), 5.55 (d, J=3.2 Hz, 1H), 5.08 (d, J=11.6Hz, 1H), 4.90 (d, J=12 Hz, 1H), 3.89 (t, J=9.6 Hz, 1H), 2.91 (m, 2H),2.56-2.17 (m, 6H), 1.84-1.72 (m, 1H), 1.55 (s, 3H), 1.17 (t, J=12.4 Hz,1H); ¹³C NMR (CDCl₃, 100 MHz): δ 169.2, 154.0, 147.7, 145.8, 138.6,134.4, 133.3, 120.6, 80.9, 71.5, 63.3, 59.9, 42.7, 36.5, 25.7, 24.3,24.1, 18.1 ppm.

Example 11. JVM 2-26

To a stirred solution of MMB (50 mg, 0.18 mmol) in dichloromethane (2mL), carbonylditriazole (46.5 mg, 0.28 mmol) was added at ambienttemperature. The reaction mixture was stirred for 10 minutes andpiperidine-1-propanol (40 mg, 0.28 mmol) was added, and the reactionmixture was maintained at ambient temperature for 30 minutes. Uponcompleted as determined by TLC, water was added and the mixtureextracted with dichloromethane. The organic layer was dried over Na₂SO₄and concentrated to afford a crude product. The crude compound waspurified by column chromatography (silica gel eluted with 3% methanol indichloromethane) to afford pure product JVM 2-26, as a white solid(yield: 81%).

¹H NMR (CDCl₃, 400 MHz): δ 6.24 (d, J=3.6 Hz, 1H), 5.74 (t, J=7.6 Hz,1H), 5.55 (d, J=3.2 Hz, 1H), 4.65 (d, J=12 Hz, 1H), 4.55 (d, J=12 Hz,1H), 4.19 (t, J=6 Hz, 2H), 3.86 (t, J=8.8 Hz, 1H), 2.88-2.82 (m, 2H),2.46-2.13 (m, 12H), 1.92 (s, 2H), 1.70-1.64 (m, 5H), 1.53 (s, 3H), 1.45(s, 2H), 1.13 (t, J=11.6 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ 169.5,155.0, 138.7, 134.5, 131.7, 120.4, 81.1, 70.3, 66.8, 63.4, 60.0, 55.5,54.5, 42.7, 36.6, 25.9, 25.5, 24.5, 24.1, 24.1, 23.9, 18.1 ppm.

Example 12. JVM 2-31

To a stirred solution of MMB (50 mg, 0.18 mmol) in dichloromethane atambient temperature, was added carbonylditriazole (46.5 mg, 0.28 mmol).The reaction mixture was stirred for 10 minutes. 6-Amino-1-hexanol (32.7mg, 0.28 mmol) was then added and the reaction was maintained for 30minutes at ambient temperature. Upon completion of the reaction, asdetermined by TLC, water was added and the mixture extracted withdichloromethane. The organic layer was dried over anhydrous Na₂SO₄ andconcentrated to afford the crude product. The crude compound waspurified by column chromatography (silica gel eluted with 3% methanol indichloromethane) to afford the pure product, JVM 2-31, as a colorlessoil (yield: 85%).

¹H NMR (CDCl₃, 400 MHz): δ 6.28 (d, J=3.6 Hz, 1H), 5.73 (t, J=8 Hz, 1H),5.59 (d, J=3.2 Hz, 1H), 4.68-4.63 (m, 2H), 4.51 (d, J=12.4 Hz, 1H), 3.90(t, J=9.2 Hz, 2H), 3.68 (t, J=6 Hz, 2H), 3.21 (d, J=6.4 Hz, 2H),2.98-2.88 (m, 2H), 2.46-2.16 (m, 6H), 1.71-1.51 (m, 8H), 1.45-1.36 (m,4H), 1.16 (t, d, J=11.6 Hz, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz): δ 169.6,156.3, 138.9, 135.7, 130.3, 120.3, 81.2, 67.2, 63.4, 62.8, 60.1, 42.7,41.0, 36.7, 32.6, 30.0, 26.5, 25.9, 25.4, 24.7, 23.9, 18.1 ppm.

Example 13. JVM 2-35

To a stirred solution of MMB (50 mg, 0.18 mmol) in dichloromethane,carbonylditriazole (46.5 mg, 0.28 mmol) was added at ambienttemperature. The reaction mixture was stirred for 10 minutes.N,N-dimethylethanolamine (24.9 mg, 0.28 mmol) was then added and thereaction was maintained for 30 minutes at ambient temperature. Uponcompletion as determined by TLC, water was added and the mixtureextracted with dichloromethane. The organic layer was dried over Na₂SO₄and concentrated to afford the crude product. The crude compound waspurified by column chromatography (silica gel eluted with 4% methanol indichloromethane) to afford the pure product, JVM 2-35 as a white solid(yield: 81%).

¹H NMR (CDCl₃, 400 MHz) δ 6.24 (d, J=3.6 Hz, 1H), 5.74 (t, J=8.4 Hz,1H), 5.55 (d, J=3.2 Hz, 1H), 4.65 (d, J=12 Hz, 1H), 4.56 (d, J=12.4 Hz,1H), 4.24 (s, 2H), 3.85 (t, J=9.6 Hz, 1H), 2.84 (d, J=9.2 Hz, 2H), 2.62(s, 2H), 2.47-2.12 (m, 12H), 1.72-1.60 (m, 1H), 1.53 (s, 3H), 1.12 (t,J=11.6 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ 169.5, 155.1, 138.7, 134.5,131.6, 120.5, 81.1, 70.4, 65.4, 63.4, 60.0, 57.6, 45.6, 42.7, 36.6,25.8, 24.4, 23.9, 18.1 ppm.

Example 14. JVM 2-40

To a stirred solution of MMB (50 mg, 0.18 mmol) in methanol (2 mL),carbonylditriazole (46.5 mg, 0.28 mmol) was added at ambienttemperature. The reaction mixture was stirred at same temperature for 1hour. Upon completion as determined by TLC, the reaction mixture wasconcentrated under reduced pressure and the crude compound was purifiedby column chromatography (silica gel eluted with 2% methanol indichloromethane) to afford pure product, JVM 2-40, as a white solid(yield: 80%).

¹H NMR (CDCl₃, 400 MHz): δ 6.24 (s, 1H), 5.72 (s, 1H), 5.53 (s, 1H),4.65 (d, t, J=11.6 Hz, 1H), 4.56 (d, J=12 Hz, 1H), 3.85 (t, J=9.6 Hz,1H), 3.77 (s, 3H), 2.83 (d, J=9.6 Hz, 2H), 2.44-2.13 (m, 6H), 1.66 (t,J=11.6 Hz, 1H), 1.52 (s, 3H), 1.12 (t, J=11.6 Hz, 1H); ¹³C NMR (CDCl₃,100 MHz): δ 169.4, 155.7, 138.7, 134.5, 131.6, 120.4, 81.1, 70.4, 63.4,60.0, 55.1, 42.7, 36.6, 25.8, 24.4, 23.9, 18.1 ppm.

Example 15. JVM 2-41

To a stirred solution of MMB (50 mg, 0.18 mmol) in dichloromethane, wasadded carbonylditriazole (46.5 mg, 0.28 mmol). The reaction mixture wasstirred at room temperature for 10 minutes, and mercaptoethanol (21.84mg, 0.28 mmol) was added. The reaction was maintained for 30 minutes.Upon completion as determined by TLC, water was added and the mixtureextracted with dichloromethane. The organic layer was dried over Na₂SO₄and concentrated to afford the crude product. The crude compound waspurified by column chromatography (silica gel eluted with 4% methanol indichloromethane) to afford the pure product, JVM 2-41, as an oil (yield:65%).

¹H NMR (CDCl₃, 400 MHz): δ 5.11 (t, J=8 Hz, 1H), 5.02 (d, J=12 Hz, 1H),4.62 (d, J=12 Hz, 1H), 3.90-3.76 (m, 4H), 3.07-3.02 (m, 3H), 2.84-2.74(m, 3H), 2.61-2.57 (m, 1H), 2.47-2.12 (m, 7H), 1.99 (s, 4H), 1.63-1.57(m, 1H), 1.53 (s, 3H), 1.11 (t, J=12.4 Hz, 1H); ¹³C NMR (CDCl₃, 100MHz): δ 175.9, 171.6, 134.7, 131.3, 81.2, 70.0, 63.3, 61.8, 60.9, 60.0,46.8, 42.7, 36.8, 36.6, 34.0, 29.7, 26.7, 24.2, 23.8, 18.0 ppm.

Example 16. JVM 2-49

To the stirred solution of MMB (70 mg, 0.27 mmol) in dichloromethane,dimethylamine (14.40 mg, 0.32 mmol) in methanol was added the reactionmixture was maintained under ambient conditions for 2 hours. Uponcompletion, the reaction mixture was concentrated to remove solvent. Thecrude reaction mixture dissolved in dichloromethane and added thethiocarbonyldiimidazole (72 mg, 0.41 mmol). The reaction mixture wasmaintained at ambient temperature for 3 hours. Upon completion, thereaction mixture was concentrated and purified by column chromatography(silica gel eluted with 3% methanol in dichloromethane) to afford pureproduct.

¹H NMR (CDCl₃, 400 MHz): δ 8.12 (s, 1H), 7.38 (s, 1H), 7.07 (s, 1H),5.51 (s, 1H), 5.19 (s, 1H), 4.32 (t, J=8 Hz, 1H), 3.80 (t, J=8 Hz, 1H),2.99-2.03 (m, 17H), 1.6 (s, 1H), 1.41 (s, 3H), 1.29-1.23 (m, 1H); ¹³CNMR (CDCl₃, 100 MHz): δ 176.5, 165.4, 144.2, 135.5, 131.1, 116.8, 115.9,79.7, 63.4, 60.0, 57.8, 53.8, 45.8, 45.3, 37.8, 30.0, 28.3, 26.1, 21.1,18.0 ppm.

Example 17. Antileukemic Activity of Various MMB Derivatives

Derivatives were screened for antileukemic activity against AML cells inculture (See FIGS. 1-7). Compounds JVM 64, JVM 66, JVM 2-26, and JVM2-49 (Examples 5, 6, 11, and 16, respectively) were the most activecompounds against AML 052308 cells in culture and were more potent thanMMB. JVM 66 (a thiocarbamate derivative of MMB, Example 6) was the mostactive molecule of this group with an LC₅₀ value of 2.6 μM, and wasabout 6-fold more cytotoxic than MMB (LC₅₀=16 μM); JVM 66 was also3-fold more potent than parthenolide (LC₅₀=7.6 μM). The derivatives JVM2-26, JVM 2-49, and JVM 64 (Examples 11, 16, and 5, respectively)exhibited similar cytotoxicity to parthenolide (LC₅₀=5, 5.2, 7.4 μM) andwere 3-fold more cytotoxic than MMB against AML 052308 cells. JVM 61(Example 4), JVM 59 (Example 3), and JVM 74 showed almost equalcytotoxicity to MMB, while JVM 57 (Example 2) and JVM 58 were lessactive than MMB. Compound JVM 88 (Example 8) was screened forantileukemic activity against the M9 ENL cell line, and against the AML123009 and AML 100510 primary isolates, exhibiting good antileukemicactivity compared to MMB in these cellular assays.

Carbamate and carbonate derivatives of MMB were also screened foractivity against M9 ENL cells at concentrations of 5, 10 and 20 μM (FIG.10). JVM 2-66, JVM 2-60, JVM 2-59, and JVM 2-49 reduced cell viabilityby greater than 50% at the lowest concentration tested. Additionally,succinic amide derivatives of MMB were screened for activity against M9ENL cells at concentrations of 5, 10 and 20 μM (FIG. 11). JVM 2-70reduced cell viability by greater than 50% at the lowest concentrationtested.

Example 18. In Vitro Growth Inhibition and Cytotoxicity

The compounds disclosed herein were also screened for anticanceractivity against a panel of 60 human tumor cell lines. The compoundswere first screened at a single concentration of 10⁻⁵ M. Compounds whichshowed more than 60% growth inhibition in at least eight human cancercell lines from the panel were selected for a complete dose-responsestudy at five concentrations: 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸M. From the initial single dose screening, 16 compounds were selectedfor a five-dose screening: JVM 2-13, JVM 2-26, JVM 2-31, JVM 2-35, JVM2-40, JVM 2-44, JVM 2-50, JVM 2-57, JVM 2-66, JVM 2-70, JVM-57 (6d),JVM-59 (6b), JVM-61 (6c), JVM-64 (6e), JVM-66, and JVM-96.

Example 19. Anti-Cancer Activity of Carbamate Derivatives ofMelampomagnolide B (MMB)

Parthenolide (PTL; 1, FIG. 8), a sesquiterpene lactone isolated from themedicinal herb Feverfew (Tanacetum parthenium), has been widely reportedin the literature as an anticancer agent that is effective against bothhematological and solid tumors.^(1,2) PTL and its analogs promoteapoptosis by inhibiting the activity of the NF-κB transcription factorcomplex, and thereby down-regulates anti-apoptotic genes under NF-κBcontrol.³ Recent studies also demonstrate that PTL induces robustapoptosis of primary acute myeloid leukemia (AML) stem cells inculture.^(4,5) AML is a clonal malignancy of the hematopoietic systemcharacterized by accumulation of immature cell populations in the bonemarrow or peripheral blood,⁶ and is the most common type of leukemia inadults but has the lowest survival rate of all leukemias.⁷

More recently, we have shown that PTL and PTL analogs also selectivelyinduce almost complete glutathione depletion and severe cell death inCD34+ AML cells,⁸ but exhibit significantly less toxicity in normalCD34+ cells. PTL analogs perturb glutathione homeostasis by amultifactorial mechanism, including inhibition of key glutathionemetabolic enzymes (GCLC and GPX1), and direct depletion of glutathione.Thus, primitive leukemia cells are uniquely sensitive to agents thattarget aberrant glutathione metabolism, an intrinsic property of primaryhuman AML cells.

PTL is a major source for several novel anti-leukemic compounds arisingfrom our research program over the past decade. The two best examplesare dimethylaminoparthenolide (DMAPT; 2, FIG. 8) and melampomagnolide B(MMB; 3, FIG. 8). MMB is a melampolide originally isolated from Magnoliagrandiflora. ⁹ MMB can be synthesized from commercially available PTLvia SeO₂/tBuOOH oxidation.^(10,11) Both of the above compounds 2 and 3have been identified as new antileukemic sesquiterpenes with propertiessimilar to PTL.^(11,12) DMAPT is currently in Phase 1 clinical studiesfor evaluation as a treatment for acute myeloid leukemia cell (AML).¹²

More importantly, from a drug design point of view, MMB is a moreintriguing molecule than either PTL or DMAPT because of the presence ofthe primary hydroxyl group at C-14, which can be structurally modifiedto improve potency, water solubility, bio-availability and tissuetargeting of the molecule.

In the current study, we have prepared a series of novel carbamateanalogs of MMB. These compounds were initially designed as potentialprodrugs of MMB. However, we have found that on examining the anticanceractivity of these compounds, several of the molecules exhibitedsignificant growth inhibition properties in a panel of sixty humancancer cell lines. Two of these compounds exhibited GI₅₀ values of 0 μMagainst the majority of the human cancer cell lines in the panel.

Carbamate analogs of MMB were prepared by reaction of thep-nitrophenyloxycarbonyl ester of MMB¹³ with a variety of primary andsecondary heterocyclic amines containing pyrrolidine, morpholine,piperidine, imidazole, triazole and pyridine moieties, to affordcarbamate products 6a-6g¹⁴ with generally improved water-solubility(Scheme 4, Table 1) compared to MMB. The key p-nitrophenyloxycarbonylester of MMB was prepared by the reaction of MMB withp-nitrophenylchloroformate in the presence of triethylamine. Allconjugation reactions were carried out at ambient temperature indichloromethane. We have reported previously that the reaction ofsesquiterpenes containing an exocyclic double bond attached to the13-position of the 5-membered lactone ring with primary and secondaryamines leads to the facile formation of Michael addition products.¹⁵However, under the reaction conditions employed in Scheme 4, the rate ofO-carbamoylation appears to be much faster than the rate of C-13 Michaeladdition, and only in a few cases, with amines such as2-morpholinoethylamine, 2-piperidinoethylamine and3-aminopropylimidazole, were Michael addition byproducts observed(usually in low yields of 5-10%), due likely to the high nucleophilicityof these amines.

All compounds were purified by column chromatography (silica gel;methanol/dichloromethane) to afford pure compounds in 50-75% yield. Thesynthesized compounds were fully characterized by ¹H NMR, ¹³C NMR andhigh resolution mass spectral analysis.¹⁴

TABLE 1 Structures, reaction conditions, yields, and melting points forcarbamate analogs of melampomagnolide B Amine Product Yield (%) Time (h)Mp (° C.)

50 12 150 6a

67 12  50

72  8 150 6c

65  6  80 6d

70  8 107 6e

75  5  70 6f

68  8  60 6g

The above carbamate analogs were evaluated for growth inhibitionproperties against a panel of 60 human cancer cell lines derived fromnine human cancer cell types, grouped into disease sub-panels thatrepresent leukemia, lung, colon, central nervous system (CNS), melanoma,renal, ovary, breast, and prostate cancer cells. Growth inhibitory(GI₅₀) effects were measured as a function of the variation of opticaldensity as a percentage of control.^(16,17) Initial screening assayswere carried out at a single concentration of 10 μM. Five analogs,6a-6e, were identified as hits based on their ability to inhibit by 60%the growth of at least 8 of the 60 tumor cell lines in the panel. Thesefive analogs were then evaluated in 5-dose assays over the concentrationrange 10⁴-10⁸ μM, and their GI₅₀ values against the tumor cell lines inthe panel determined (Table 2). Two analogs, 6a and 6e, were identifiedas lead compounds and generally exhibited improved growth inhibitionagainst all human tumor cell lines when compared to PTL (1) and DMAPT(2), with the exception of the leukemia cell line subpanel; in thesecell lines, the GI₅₀ values for DMAPT compared very favorably with thosefor both 6a and 6e. Compound 6a exhibited potency against leukemia cellline CCRF-CEM, melanoma cell line MDA-MB-435 and breast cancer cell lineMDA-MB-468 in the nanomolar range with GI₅₀ values of 680, 460 and 570nM, respectively. Compound 6e was found to possess potent anti-leukemicactivity against leukemia cell line CCRF-CEM, non-small cell lung cancercell line HOP-92 and renal cancer cell line RXF 393 with GI₅₀ values of620, 650 and 900 nM, respectively, (Table 2).

Compounds 6a and 6e also exhibited significant growth inhibition againstthe following sub-panels of human cancer cell lines: non-small cell lungcancer (GI₅₀ values 0.65-1.45 μM); colon cancer (GI₅₀ values 1.12-2.06μM); melanoma (GI₅₀ values 0.46-2.84 μM); renal cancer _((GI50) values0.90-2.60 μM); and breast cancer (GI₅₀ values 0.57-3.07 μM) (Table 2).

We have determined the hydrolytic stability of the above five carbamatederivatives in human plasma and have shown that compounds 6b-6e havehalf-lives in the range 100-180 min, while compound 6a has a much longerhalf-life of 8 h in human plasma. Thus, we consider compounds 6b-6d tobe anticancer agents that are also metabolized by plasma esterases tothe active parent compound MMB, while compound 6a would be considered amore potent anticancer agent than MMB that is likely not metabolized toMMB in vivo.

The above results are interesting for a number of reasons: first, theantileukemic activities of 6a and 6e against the sub-panel of humanleukemia cells indicates that these carbamate analogs of MMB are morepotent than the parent compound. Second, the potent growth inhibition ofhuman solid tumor cell lines by 6a and 6e is the first report of suchactivities for MMB analogs.

Third, these interesting results indicate that structural modificationof the MMB molecule through appropriate carbamoylation of the primaryhydroxyl group can lead to an improvement in the anticancer propertiesof MMB.

The above MMB analogs, like PTL and DMAPT, are inhibitors of the NFκBpathway, activators of the nuclear kinase JNK, and selectively depleteglutathione levels in hematopoietic cancer stem cells, leading to anincrease in reactive oxygen species (ROS) and subsequent apoptosis (FIG.9).^(4,8,11) We have recently shown that hematopoietic cancer stem cellshave lower levels of reduced glutathione (GSH) and increased levels ofoxidized glutathione (GSSG) when compared to normal stem cells, and arethus more vulnerable to agents such PTL and MMB and its analogs thatinduce oxidative stress through generation of ROS.¹¹ Specifically, PTLand MMB analogs inhibit several crucial enzymes in the glutathionepathway (i.e., GCLC and GPX1) leading to severe depletion of cellularglutathione and resulting in oxidative stress and apoptosis.

In summary, we have reported on a series of novel carbamate derivativesof MMB derived from heterocyclic and heteroaromatic amines. Among thesederivatives, compounds 6a and 6e have been identified as potentanticancer agents with growth inhibition activities in the nanomolarrange against a variety of hematological and solid tumor cell lines.Analogs 6a and 6e exhibit promising anti-leukemic activity against humanleukemia cell line CCRF-CEM with GI₅₀ values of 680 and 620 nM,respectively. Compound 6a also exhibits GI₅₀ values of 460 and 570 nMagainst MDA-MB-435 melanoma and MDA-MB-468 breast cancer cell lines,respectively, and 6e has GI₅₀ values of 650 and 900 nM against HOP-92non-small cell lung and RXF 393 renal cancer cell lines, respectively.Further structure-activity relationship studies will focus on thestructural optimization of these interesting lead analogs and on themolecular basis for their mechanism of action.

TABLE 2 Growth inhibition (GI₅₀; μM)^(b) data for PTL (1), DMAPT (2) andcarbamoylated MMB analogs 6a-6e against a panel of human cancer celllines 1a^(a) 2a^(a) 6a 6b 6c 6d 6e Panel/cell line GI₅₀ GI₅₀ GI₅₀ GI₅₀GI₅₀ GI₅₀ GI₅₀ Leukemia CCRF-CEM 7.94 1.99 0.68 2.49 2.65 3.03 0.62HL-60(TB) 5.01 1.58 2.04 4.15 ND 3.59 ND K-562 19.9 2.51 3.45 3.26 ND3.37 ND MOLT-4 15.8 3.16 2.05 3.48 5.54 5.00 2.32 RPMI-8226 7.94 2.511.98 8.71 8.20 5.72 2.57 SR ND^(c) ND 1.38 10.2 4.10 3.65 2.36 Non-smallcell lung cancer HOP-92 12.5 10.0 1.45 2.25 2.25 2.30 0.65 NCI-H522 5.012.51 1.25 1.78 1.64 2.13 1.26 Colon cancer COLO 205 15.8 31.6 2.06 4.783.54 8.89 1.79 HCT-116 10.0 5.01 1.41 3.18 1.89 2.87 1.13 SW-620 15.83.98 1.46 3.46 3.13 3.48 1.12 CNS cancer SF-539 19.9 2.51 1.98 15.0 5.7714.4 1.76 SNB-75 50.1 ND 6.13 18.0 3.78 19.5 1.71 Melanoma LOX IMVI 7.9410.0 2.23 7.88 4.96 4.84 1.95 MALME-3M 12.5 ND 1.90 4.18 7.52 6.12 2.32M14 ND 15.8 2.84 9.65 5.58 7.97 1.59 MDA-MB-435 ND 7.94 0.46 6.56 6.015.89 2.24 Ovarian cancer IGROV1 19.9 19.9 2.31 4.09 14.4 3.49 3.66OVCAR-3 19.9 12.5 1.69 8.41 ND 6.20 ND Renal cancer ACHN ND 15.8 1.793.80 2.75 3.74 1.75 CAKI-1 10.0 12.5 2.03 6.86 2.88 4.31 1.99 RXF 39312.5 15.8 1.20 4.08 2.22 3.00 0.90 TK-10 ND 3.16 2.60 3.11 3.78 3.932.51 Prostate cancer DU-145 ND 5.01 2.44 7.42 4.59 3.74 3.49 Breastcancer MCF7 15.8 5.01 1.62 3.91 2.85 3.54 1.33 BT-549 ND 5.01 2.69 4.752.60 4.99 1.47 T-47D ND 39.8 3.07 4.86 6.19 6.32 2.23 MDA-MB-468 ND ND0.57 2.30 3.22 3.26 1.29 GI₅₀ values <1 μM are bolded. ^(a)GI₅₀ valuesobtained from NCI database. ^(b)GI₅₀, concentration of analog (μM) thathalves cellular growth. ^(c)ND not determined.

REFERENCES AND NOTES FOR EXAMPLE 19

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Phytochemistry 1984, 23, 2372.-   10. Macias, F. A.; Galindo, J. C. G.; Massanet, G. M. Phytochemistry    1992, 31, 1969.-   11. Shama, N.; ShanShan, P.; Fred, K. H.; Craig, T. J.; Peter, A. C.    Bioorg. Med. Chem. 2011, 19, 1515.-   12. Guzman, M. L.; Rossi, R. M.; Neelakantan, S.; Li, X.;    Corbett, C. A.; Hassane, D. C.; Becker, M. W.; Bennett, J. M.;    Sullivan, E.; Lachowicz, J. L.; Vaughan, A.; Sweeney, C. J.;    Matthews, W.; Carroll, M.; Liesveld, J. L.; Crooks, P. A.;    Jordan, C. T. Blood 2007, 110, 4427.-   13. Synthetic procedure and analytical data for the    p-nitrophenyloxycarbonyl ester of MMB (5): To the reaction mixture    of MMB (100 mg, 0.378 mmol) and triethylamine (45.8 mg, 0.454 mmol)    in dichloromethane (2 mL), p-nitrophenylchloroformate (76.3 mg,    0.378 mmol) was added at 0° C. The reaction mixture was stirred for    24 h at ambient temperature. When the reaction was completed    (monitored by TLC), water was added to the reaction mixture and the    aqueous mixture was extracted with dichloromethane. The organic    layer was washed with water, followed by brine solution, dried over    anhydrous Na2SO4 and concentrated to afford the crude product. The    crude product was purified by column chromatography (silica gel, 2%    methanol in dichloromethane) to afford compound 5 as a pale yellow    solid. ¹H NMR (CDCl3, 400 MHz): o 8.26 (d, J=9.6 Hz, 2H), 7.37 (d,    J=9.8 Hz, 2H), 6.25 (s, 1H), 5.83 (t, J=8.4 Hz, 1H), 5.56 (s, 1H),    4.81 (d, J=12.8 Hz, 1H), 4.72 (d, J=12.4 Hz, 1H), 3.86 (m, 1H), 2.85    (m, 2H), 2.56 (m, 7H), 1.77 (m, 2H), 1.55 (s, 1H), 1.16 (t, J=13.2    Hz, 1H). ¹³C NMR (CDCl3, 100 MHz): o 169.1, 155.2, 152.2, 145.4,    138.6, 133.6, 132.8, 125.3, 121.5, 120.3, 80.8, 71.5, 63.1, 59.8,    42.7, 36.4, 25.6, 24.9, 23.9, 17.9 ppm.-   14. General synthetic procedure and analytical data for carbamate    derivatives of MMB: To the p-nitrophenyloxycarbonyl ester derivative    of MMB (5) (70 mg, 0.16 mmol) in dichloromethane (2 mL), the    appropriate amine (0.16 mmol) was added at 0° C. The reaction    mixture was stirred for 18 h at ambient temperature. When the    reaction was completed (monitored by TLC), water was added to the    reaction mixture and the aqueous mixture was extracted with    dichloromethane. The organic layer was washed with water, followed    by brine solution, dried over anhydrous Na₂SO₄ and concentrated to    afford the crude product. The crude product was purified by column    chromatography (silica gel, 5% methanol in dichloromethane) to    afford the carbamate analogs (6a-g) as white solids.    ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-5-yl)methyl-4,4-difluoropiperidine-1-carboxylate    (6a): ¹H NMR (CDCl₃), 400 MHz): o 6.27 (d, J=2.8 Hz, 1H), 5.67 (t,    J=8.4 Hz, 1H), 5.56 (s, 1H), 4.69 (d, J=12.4 Hz, 1H), 4.52 (d, J=12    Hz, 1H), 3, 87 (t, J=9.6 Hz, 1H), 3.60 (br s, 4H), 2.87 (d, J=9.2    Hz, 2H), 2.50-2.16 (m, 6H), 1.96 (br s, 4H), 1.71 (t, J=10 Hz, 1H),    1.55 (s, 3H), 1.14 (t, J=12 Hz, 1H). ¹³C NMR (CDCl3, 100 MHz): o    169.4, 154.8, 138.7, 135.3, 129.9, 121.5, 120.5 (t, JCF=5.3 Hz, 1C),    81.1, 67.7, 63.4, 60.0, 42.7, 41.0, 36.7, 34.0, 25.8, 24.4, 23.9,    18.1 ppm. HRMS (ESI) m/z calcd for C₂₁H₂₈F₂NO₅ (M+H)⁺ 412.1930.    found 412.1933.    ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-5-yl)methyl-(2-(pyrrolidin-1-yl)ethyl)carbamate    (6b): ¹H NMR (CDCl3, 400 MHz): o 6.22 (d, J=3.2 Hz, 1H), 5.65 (t,    J=7 Hz, 1H), 5.55 (s, 1H), 4.62 (d, J=11.6 Hz, 1H), 4.47 (d, J=12.4    Hz, 1H), 3.82 (t, J=9.6 Hz, 1H), 3.35 (s, 2H), 2.90-2.83 (m, 2H),    2.70 (s, 4H), 2.42 (d, J=9.6 Hz, 2H), 2.38-2.13 (m, 7H) 1.84 (s,    4H), 1.66 (t, J=12 Hz, 1H), 1.52 (s, 3H), 1.13 (t, J=11.6 Hz, 1H).    ¹³C NMR (CDCl₃, 100 MHz): δ 169.3, 156.1, 138.6, 135.3, 129.8,    120.1, 80.9, 67.0, 63.1, 59.8, 55.1, 53.8, 42.5, 38.9, 36.5, 25.6,    24.3, 23.6, 23.2, 17.8 ppm. HRMS (ESI) m/z calcd for C₂₂H₃₃N₂O₅    (M+H)⁺ 405.2384. found 405.2390.    ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-5-yl)methyl-(2-(pyridin-2-yl)ethyl)carbamate    (6c): ¹H NMR (CDCl₃, 400 MHz): δ 8.50 (s, 1H), 7.62 (t, J=7.2 Hz,    1H), 7.15 (d, J=7.6 Hz, 2H), 6.19 (s, 1H), 5.64 (t, J=8 Hz, 1H),    5.56-5.56 (m, 2H), 4.62 (d, J=12.4 Hz, 1H), 4.44 (d, J=12.8 Hz, 1H),    3.84 (t, J=9.2 Hz, 1H), 3.60 (d, J=6 Hz, 2H), 2.99-2.82 (m, 4H),    2.41-2.12 (m, 5H), 1.75 (s, 1H), 1.64 (d, J=10.4 Hz, 1H), 1.52 (s,    3H), 1.07 (t, J=13.6 Hz, 1H). ¹³C NMR (CDCl3, 100 MHz): o 169.3,    159.1, 156.0, 149.1, 138.6, 136.6, 135.5, 129.9, 123.4, 121.6,    120.2, 81.0, 67.0, 63.2, 59.8, 42.5, 40.1, 37.1, 36.6, 25.8, 24.4,    23.7, 17.9 ppm. HRMS (ESI) m/z calcd for C₂₃H₂₉N₂O₅ (M+H)⁺ 413.2071.    found 413.2073.    ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-5-yl)-methyl(2-morpholino    ethyl) carbamate (6d): ¹H NMR (CDCl₃, 400 MHz): δ 6.11 (d, J=3.6 Hz,    1H), 5.58 (t, J=8 Hz, 1H), 5.43 (d, J=2.8 Hz, 1H), 5.06 (s, 1H),    4.53 (d, J=12.4 Hz, 1H), 4.34 (d, J=12 Hz, 1H), 3.74 (t, J=8.8 Hz,    1H), 3.56 (s, 4H), 3.16 (t, J=5.2 Hz, 2H), 2.78-2.72 (m, 2H), 2.32    (s, 8H), 2.21-2.00 (m, 4H), 1.55 (t, J=10.4 Hz, 1H), 1.41 (s, 3H),    1.01 (t, J=12 Hz, 1H). ¹³C NMR (CDCl3, 100 MHz): δ 169.2, 155.9,    138.7, 135.4, 130.2, 120.0, 80.9, 67.0, 66.7, 63.1, 59.8, 57.2,    53.1, 42.5, 37.0, 36.5, 25.7, 24.4, 23.7, 17.8 ppm. HRMS (ESI) m/z    calcd for C₂₂H₃₃N₂O₆ (M+H)⁺ 421.2333. found 421.2331.    ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-5-yl)methyl-(5-(methylthio)-1H-1,2,4-triazol-3-yl)carbamate    (6e): ¹H NMR (CDCl3, 400 MHz): δ 6.22 (s, 2H), 5.82 (t, J=8 Hz, 1H),    5.50 (s, 1H), 4.90 (d, J=12.4 Hz, 1H), 4.81 (d, J=12.4 Hz, 1H), 3.85    (t, J=9.6 Hz, 1H), 2.95 (s, 1H), 2.85 (d, J=9.2 Hz, 1H), 2.48-2.15    (m, 8H), 1.70-1.53 (m, 6H), 1.13 (t, J=12.4 Hz, 1H) ppm. ¹³C NMR    (CDCl3, 100 MHz): δ 169.1, 163.1, 157.4, 149.9, 138.5, 133.3, 132.7,    120.2, 80.7, 70.1, 62.9, 59.7, 42.4, 36.2, 25.6, 24.3, 23.7, 17.8,    13.5 ppm. HRMS (ESI) m/z calcd for C₁₉H₂₅N₄O₅S (M+H)⁺ 421.1540.    found 421.1524.    ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-5-yl)methyl-(3-(1H-imidazol-1-yl)-propyl)carbamate    (6f): ¹H NMR (CDCl₃, 400 MHz): δ 7.56 (s, 1H), 7.09 (s, 1H), 6.95    (s, 1H), 6.26 (d, J=3.6 Hz, 1H), 5.69 (t, J=8 Hz, 1H), 5.56 (d,    J=3.2 Hz, 1H), 4.85 (s, 1H), 4.64 (d, J=12.4 Hz, 1H), 4.51 (d,    J=12.4 Hz, 1H), 4.04 (t, J=7.2 Hz, 2H), 3.88 (t, J=9.2 Hz, 1H), 3.21    (d, J=6 Hz, 2H), 2.93-2.85 (m, 2H), 2.47-2.16 (m, 7H), 2.03 (t,    J=6.4 Hz, 1H) 1.70 (t, J=10.8 Hz, 1H), 1.55 (s, 3H), 1.15 (t, J=12.4    Hz, 1H). ¹³C NMR (CDCl3, 100 MHz): δ 169.5, 156.3, 138.9, 137.1,    135.3, 130.4, 129.6, 120.2, 118.8, 81.1, 67.3, 63.3, 60.0, 44.4,    42.7, 38.3, 36.7, 31.5, 25.8, 24.6, 23.8, 18.0 ppm. HRMS (ESI) m/z    calcd for C₂₂H₃₀N₃O₅ (M+H)⁺ 416.2180. found 416.2183.    ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]-cyclodeca[1,2-b]furan-5-yl)methyl-(3-morpholinopropyl)carbamate    (6g)¹H NMR (CDCl3, 400 MHz): δ 6.23 (s, 1H), 5.74 (s, 1H), 5.65 (t,    J=8 Hz, 1H), 5.54 (s, 1H), 4.58 (d, J=12 Hz, 1H), 4.48 (d, J=16 Hz,    1H), 3.85 (t, J=9.2 Hz, 1H), 3.72 (s, 4H), 3.27 (s, 2H), 2.89-2.83    (m, 2H), 2.46-2.11 (m, 12H), 1.69 (t, J=12 Hz 3H), 1.52 (s, 3H),    1.11 (t, J=12 Hz, 1H). ¹³C NMR (CDCl₃, 100 MHz): δ 169.5, 156.3,    138.9, 135.7, 129.9, 120.4, 81.2, 67.0, 66.9, 63.4, 60.1, 57.4,    53.6, 42.7, 36.8, 25.9, 25.5, 24.6, 23.9, 18.1 ppm. HRMS (ESI) m/z    calcd for C₂₃H₃₅N₂O₆ (M+H)⁺ 435.2490. found 435.2482.-   15. Neelakantan, S.; Shama, N.; Guzman, M. L.; Jordan, C. T.;    Crooks, P. A. Bioorg. Med. Chem. Lett. 2009, 19, 4346.-   16. Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91.-   17. Acton, E. M.; Narayanan, V. L.; Risbood, P. A.; Shoemaker, R.    H.; Vistica, D. T.; Boyd, M. R. J. Med. Chem. 1994, 37, 2185.

Example 20. Efficacy of Ester and Amide Derivates of MMB AgainstLeukemia Cell Lines

Ester and amide derivatives of melampomagnolide B were screened foranti-leukemic activity against M9ENL AML cell lines (FIG. 12; FIG. 14;FIG. 15; FIG. 16). For each compound, in vitro cell culture studies wereperformed in which a range of concentrations (0.25-20 μM) was tested forcytotoxicity against leukemia cell lines, primary leukemia specimens andnormal bone marrow controls. Evaluations were performed generally after24 hours of drug exposure using viability labeling with trypan blue dye,as well as flow cytometric labeling with Annexin V and propidium iodideto delineate dead cell populations. For all studies, native parthenolide(PTL) was included as a reference control. As expected, relativecytotoxicity varied considerably as a function of the specificstructural modifications made to each molecule.

Compounds BS-1-28, BS-2-01, BS-2-05 and BS-2-31, BS-2-32 were the mostactive compounds against AML 052308 cells in culture and were morepotent than the parent compound, MMB. BS-2-04 (the indole acrylic acidderivative of MMB) was the most active molecule with an EC₅₀ value of0.72 μM, and was about 22-fold more cytotoxic than MMB (EC₅₀=16 μM);BS-2-04 was also 10-fold more potent than parthenolide (EC₅₀=7.6 μM).The analogs BS-1-28, BS-2-05, BS-2-31, and BS-2-32 exhibited around3-fold more potency (EC₅₀=2.2, 2.5, 3.0, 4.0 μM) in comparison withparthenolide, and were around 7-fold more cytotoxic than MMB against AML052308 cells. BS-1-98 showed almost equal cytotoxicity to the parentcompound, MMB.

Compound BS-2-04 was screened for anti-leukemic activity against the M9ENL cell line, and AML 123009 and AML 100510 primary isolates, andexhibited good anti-leukemic activity compared to parent compound MMB inthese cellular assays. Moreover, other compounds consistentlydemonstrated greater cytotoxicity than PTL (FIG. 12; FIG. 14). The EC₅₀values of certain compounds were consistently approximately 10-foldergreater than PTL. EC₅₀ values of these compounds are 0.27 μM (BS-2-04),2.2 μM (BS-2-01), 2.5 μM (BS-2-05), 3.0 μM (BS-2-31), 4.0 μM (BS-2-32),2.9 μM (JVM 3-38) against M9ENL AML cell lines. EC₅₀ values of othercompounds were: 11 μM (JVM 3-39), 4.7 μM (JVM 3-22), 3.3 μM (JVM 3-30),11 μM (JVM 340), 2.9 μM (JVM 3-38), 1.7 μM (JVM 3-36), 13 μM (JVM 3-41)and 3.5 μM (JVM 3-44) against M9ENL AML cell lines. Amide derivatives ofMMB afforded EC₅₀ values of 3.5 μM (JVM 3-50), 0.016 μM (JVM 3-53), 5.9μM (JVM 3-57), 12 μM (JVM 3-51), 8.3 μM (JVM 3-52) and 28 μM (JVM 3-46).Compound JVM 3-53 exhibited almost 30-fold greater cytotoxicity thanPTL, and compound JVM 3-36 was 3-fold more potent than PTL. Theremaining compounds had activities similar to PTL (FIG. 15; FIG. 16).

Example 21. Efficacy of Amide and Ester Derivatives of MMB Against aPanel of 60 Human Tumor Cell Lines

MMB analogs were also screened for anticancer activity against a panelof 60 human tumor cell lines. The compounds were first screened at asingle concentration of 10⁻⁵ M. Compounds which showed more than 60%growth inhibition in at least eight human cancer cell lines from thepanel of sixty cell lines were selected for a complete dose-responsestudy at five different concentrations of drug (10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M,10⁻⁷ M and 10⁻⁸ M). From the initial single dose screen, 5 compounds(BS-1-28, 2-01, 2-30, 2-65, 2-68) were selected for five dose screening.Compound BS-1-28 showed remarkable anticancer activity against melanoma,leukemia, colon, CNS, breast, ovarian, non-small cell lung, renal, andprostate cancers (FIG. 17A-I). Compound BS-1-28 was found to be a verypotent analogue with GI₅₀ values <10 nM against BT-549 and T-467 Dbreast cancer cell lines, and this compound also exhibited potentnanomolar growth inhibition against CCRF-CEM, HL-60 (TB), K-562 andRPMI-8226 leukemia cell lines; HOP-92 and NCI-H522 non-small cell lungcancer cell lines; HCT-116, HCT-15 and SW-620 colon cancer cell lines;LOX IMVI, M14 and MDA-MB-435 melanoma cell lines; and 786-0, ACHN andUO-31 renal cancer cell lines (FIG. 17A-I). Five dose screening studiesfor other compounds are currently in progress.

The compounds JVM 4-14 and JVM 4-19 were screened at a singleconcentration of 10⁻⁵ M. JVM 4-14 showed potent activity against allleukemia lines tested as well as some NSCLC, colon cancer, melanoma,renal cancer and breast cancer cell lines (FIG. 19A-B). JVM 4-14 alsoshowed moderate activity against CNS cancer and ovarian cancer.Interestingly, JVM 4-19 did not show potent activity against leukemiacell lines, but instead showed potent activity against the majority ofcolon cancer, melanoma, renal cancer, prostate cancer and breast cancercell lines tested (FIG. 20A-B). JVM 4-19 also had moderate activityagainst NSCLC, CNS cancer and ovarian cancer cell lines.

In summary, novel ester and amide conjugates of MMB have been designedand synthesized utilizing a variety of substituted organic carboxylicacids and acid chlorides, a variety of heterocyclic carboxylic acids,and a series of aromatic and aliphatic carboxylic acids in the presenceof standard EDCI coupling conditions. The newly synthesized compoundshave been evaluated for their anti-leukemic activity against two acutemyeloid leukemia cell lines (AML 052308, M9 ENL) and 2 primary AMLisolates (AML 100510, and AML 123009). The indole ester conjugate ofMMB, BS-2-04, was found to be the most potent compound against AML052308 cells with an EC₅₀ value of 0.72 μM. Other MMB derivatives(BS-1-28, BS-2-01, BS-2-32 and BS-2-05) showed greater cytotoxicity thanthe parent compound, MMB (EC₅₀=16 μM), against the above cultured AMLcells.

Compound BS-1-28 was also found to be a very potent analogue with GI₅₀values <10 nM against BT-549 and T-467 D breast cancer cell lines, andthis compound also exhibited potent growth inhibition (GI₅₀=288 nM)against CCRF-CEM leukemia cell lines in the NCI panel of 60 human cancercell lines (FIG. 18; Table 3).

TABLE 3 NCI five dose result for BS-1-28 Log10 Concentration Time MeanOptical Densities Percent Growth Panel/Cell Zero Ctrl −8.0 −7.0 −6.0−5.0 −4.0 −8.0 −7.0 −6.0 −5.0 −4.0 GI50 TGI LC50 Leukemia CCRF-CEM 0.4672.683 2.658 2.460 0.533 0.395 0.224 99 90 3 −15 −37  2.86E−71.45E−6 >1.00E−4 HL-60(TB) 0.702 2.456 2.694 2.663 1.193 0.581 0.300 114112 28 −17 −57  5.46E−7 4.15E−6  6.58E−5 K-562 0.202 1.558 1.598 1.5700.269 0.199 0.154 100 98 5 −1 −24  3.27E−7 5.79E−6 >1.00E−4 MOLT-4 0.6032.579 2.870 2.641 1.820 0.583 0.413 105 103 62 −3 −32  1.51E−68.89E−6 >1.00E−4 RPMI-8226 0.696 2.425 2.371 2.253 0.638 0.514 0.462 9792 −8 −26 −34  2.64E−7 8.27E−7 >1.00E−4 SR 0.509 2.005 2.032 1.975 1.2970.368 0.309 162 98 53 −28 −39  1.08E−6 4.51E−6 >1.00E−4 Non-Small Cell  Lung Cancer A549/ATCC 0.518 2.528 2.351 2.454 2.362 0.305 0.246 91 96 92−41 −53  2.06E−6 4.90E−6  5.93E−5 HOP-92 1.360 1.823 1.704 1.688 1.5470.120 0.038 74 71 40 −91 −97  4.80E−7 2.02E−6  4.66E−6 NCI-H226 1.1082.794 2.654 2.595 2.384 0.303 0.327 92 88 74 −73 −71  1.47E−6 3.21E−6 7.01E−6 NCI-H23 0.480 1.444 1.419 1.410 1.297 0.158 0.075 96 96 85 −67−84  1.69E−6 3.62E−6  7.72E−6 NCI-H322M 0.727 1.832 1.623 1.784 1.7020.129 0.079 81 95 88 −82 −89  1.68E−6 3.29E−6  6.47E−6 NCI-H460 0.2472.228 2.347 2.253 1.958 0.682 0.061 166 101 86 −67 −75  1.73E−6 3.66E−6 7.75E−6 NCI-H522 0.874 1.733 1.589 1.774 1.239 0.269 0.246 83 104 43−69 −72  7.57E−7 2.40E−6  6.72E−6 Colon Cancer     COLO 205 0.471 1.5950.773 1.150 0.906 0.183 0.474 27 60 39 −61 −63   2.44E−6  7.730-6HCC-2998 0.724 2.399 2.446 2.473 2.467 0.295 9.114 103 104 104 −59 −84 2.14E−6 4.33E−6  8.77E−6 HCT-116 0.282 1.699 1.653 1.645 0.108 0.0170.026 97 96 −62 −94 −91  1.96E−7 4.06E−7  8.41E−7 HCT-15 0.282 1.7581.660 1.667 0.166 0.102 0.651 93 94 −41 −64 −82  2.11E−7 4.95E−7 2.43E−6 HT29 0.194 0.934 0.955 1.002 0.146 0.083 0.040 103 109 −25 −57−80  2.76E−7 6.51E−7  5.97E−4 KM12 0.542 2.550 2.413 2.472 2.390 0.3430.098 93 96 92 −42 −82  2.05E−6 4.84E−6  1.56E−5 SW-620 0.272 1.8491.741 1.811 0.221 0.091 0.067 93 98 −19 −67 −75  2.56E−7 8.88E−7 4.47E−6 CNS Cancer     SF-268 0.559 1.876 1.649 1.823 1.654 0.108 0.10798 96 83 −81 −85  1.59E−6 3.22E−6  6.50E−8 SF-295 0.685 2.629 2.3412.411 2.227 0.369 0.035 85 89 79 .46 −95  1.71E−6 4.28E−6  1.20E−5SF-539 0.726 1.866 1.752 1.788 1.520 0.062 0.089 90 93 70 −91 −91 1.32E−6 2.71E−4  6.53E−6 SNB-19 0.467 1.820 1.776 1.718 4.562 0.1380.166 57 92 81 −70 −64  1.60E−6 3.93E−6  7.33E−6 SNB-75 0.725 1.5351.308 1.352 1.133 0.011 0.013 72 77 50 −98 −58  1.01E−6 2.19E−6  4.72E−6U251 0.544 2.552 2.519 2.469 2.391 0.222 0.157 98 96 92 −59 −74  1.69E−64.06E−6  8.68E−6 Melanoma     LOX IMVI 0.192 1.520 1.438 1.432 0.0530.023 0.037 93 93 −72 −88 −81  1.82E−7 3.65E−7  7.32E−7 MALME-3M 0.6771.241 1.193 1.190 1.142 0.090 0.122 91 91 82 −87 −82  1.56E−6 3.07E−6 6.07E−6 M14 0.494 1.525 1.398 1.393 0.972 0.097 0.041 88 87 46 −80 −92 8.14E−7 2.32E−6  5.76E−6 MDA-MB-435 0.452 2.211 2.161 2.175 1.220 0.0930.020 97 98 44 −76 −96  7.63E−7 2.28E−6  5.85E−6 SK-MEL-2 1.110 2.0382.064 2.158 2.004 0.362 0.153 103 113 96 −67 −88  1.92E6 3.88E−6 7.83E−6 SK-MEL-28 0.692 1.900 1.778 1.915 4.500 0.090 0.046 90 101 6747 −93  1.28E−6 2.72E−6  5.74E−6 SK-MEL-5 0.665 2.523 2.727 2.657 2.4040.014 0.035 96 92 67 −98 −95  1.26E−6 2.54E−6  5.12E−6 UACC-257 1.1392.637 2.606 2.535 2.377 0.290 0.246 98 93 53 −75 −78  1.61E−6 3.36E−6 6.98E−6 UACC-62 0.675 2.350 2.294 2.198 1.930 0.162 0.166 97 91 75 −73−15  1.47E−6 3.21E−6  6.98E−6 Ovarian Cancer     OVCAR-3 0.484 1.4600.670 0.973 0.413 0.039 0.023 19 50 −15 −92 −95   5.92E−7 2.66E−6OVCAR-4 0.649 1.385 0.678 1.299 1.004 0.274 0.105 4 88 48 −58 −84  2.85E−6 8.43E−6 OVCAR-5 0.561 1.603 1.444 1.561 1.149 0.228 0.069 85 9657 −59 −88  1.14E−6 3.07E−6  8.22E−6 OVCAR-8 0.546 2.552 1.719 2.2071.932 0.648 0.203 58 63 69 5 −63  1.99E−6 1.19E−4  6.46E−5 NCl/ADR-0.509 1.708 1.695 1.712 4.434 0.499 0.291 99 100 77 −2 −43  2.20E−69.42E−6 >1.00E−4 RES   SK-OV-3 0.932 1.391 1.309 1.347 1.288 0.491 0.13482 90 77 −47 −86  1.65E−6 4.47E−6  1.17E−5 Renal Cancer     786-0 0.7322.126 2.075 2.149 0.884 0.073 0.045 96 102 11 −90 −94  3.71E−7 1.28E−6 9.01E−6 A498 1.215 2.022 2.105 2.037 2.000 0.012 0.065 110 102 97 −99−95  1.74E−6 3.43E−6  5.63E−6 ACHN 0.576 1.704 1.429 1.540 0.792 0.0490.030 76 55 19 −91 −95  3.42E−7 1.49E−6  4.22E−6 CAKI-1 0.575 2.7622.466 2.539 1.764 0.007 −0.001 86 90 55 −99 −100  1.08E−6 2.18E−6 4.82E−6 RXF 393 0.893 1.288 1.129 1.201 1.012 0.161 0.245 73 85 54 −77−65  1.07E−6 2.58E−6  6.23E−6 SN12C 0.727 2.414 1.743 1.918 2.025 0.4690.296 60 71 77 −35 −59  1.14E−6 4.83E−6  9.05E−5 TK-10 0.969 1.838 1.7981.983 1.613 0.044 0.024 95 117 74 −96 −98  1.39E−6 2.73E−6  5.39E−6UO-31 0.802 2.408 1.965 1.990 1.584 0.061 0.092 72 74 49 −92 −89 8.87E−4 2.21E−6  5.00E−6 Prostate Cancer     PC-3 0.614 2.481 2.2122.234 1.075 0.130 0.039 88 87 73 −79 −94  1.42E−6 3.62E−4  6.45E−6DU-145 0.370 1.647 1.530 1.618 1.179 0.019 0.009 91 98 63 −95 −98 1.21E−6 2.51E−6  5.20E−6 Breast Cancer     MCF7 0.348 1.951 1.365 1.7320.517 0.130 0.154 63 88 11 −63 −56  3.02E−7 1.39E−6  6.69E−6 MDA-MB-0.538 1.667 1.546 1.515 1.174 0.228 0.193 89 87 56 −58 −73  1.14E−63.12E−6  8.56E−6 231/ATCC HS 57BT 1.120 2.299 1.676 2.106 2.035 1.0650.901 47 84 78 −5 −20 8.71E−6 >1.00E−4 BT-549 1.331 1.928 0.926 1.4171.034 0.159 0.019 −30 14 −22 −88 −99 <1.00E−8  2.64E−6 T-47D 0.637 1.3680.523 0.930 0.897 0.362 0.428 −2 40 35 −43 −33 <1.00E−8 >1.00E−4MDA-MB-468 0.761 1.596 1.396 1.550 0.787 0.219 0.286 76 95 3 −71 −52 3.07E−7 1.10E−6  5.17E−6

Example 22.(1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-N-(5-(methylthio)-1H-1,2,4-triazol-3-yl)-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-5-carboxamide(JVM 3-53)

To melampomagnolic acid (30 mg, 0.107 mmol in dichloromethane, was addedEDC (30 mg, 0.161 mmol), HOBt (21.7 mg, 0.161 mmol), triethylamine (32.6mg, 0.323 mmol) and 5-(methylthio)-1H-1,2,4-triazol-3-amine (13.9 mg,0.107 mmol) at ambient temperature. The reaction mixture was stirred for3 h at ambient temperature. When the reaction was complete (monitored byTLC), water was added to the reaction mixture and the aqueous mixturewas extracted with dichloromethane. The organic layer was washed withwater, followed by brine solution, dried over anhydrous Na₂SO₄ andconcentrated to afford the crude product. The crude product was purifiedby column chromatography (silica gel, 3% methanol in dichloromethane) toafford compound JVM 3-53 as a white solid (yield: 51%).

NMR (CDCl₃, 400 MHz): δ 6.58 (t, J=8 Hz, 1H), 6.34 (s, 2H), 6.20 (d,J=3.2 Hz, 1H), 5.44 (d, J=3.2 Hz, 1H), 3.83 (t, J=9.6 Hz, 1H), 3.28 (d,J=9.2 Hz, 1H), 3.01 (dd, J=15.2, 6.4 Hz, 1H), 2.91-2.85 (m, 1H),2.54-2.26 (m, 8H), 1.70-1.63 (m, 1H), 1.58 (s, 3H), 1.29-1.2 (m, 1H).¹³C NMR (CDCl₃, 100 MHz): δ 169.3, 168.6, 163.4, 158.0, 141.8, 138.9,134.9, 120.4, 81.2, 62.4, 59.6, 42.5, 36.1, 25.6, 24.6, 24.4, 18.1, 13.9ppm.

Example 23.((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2′,3′:9,10]cycodeca[1,2-b]furan-5-yl)methyl2-naphthoate (JVM 3-36)

To MMB (50 mg, 0.189 mmol) in dichloromethane (2 mL), was added2-naphthoyl chloride (35.9 mg, 0.189 mmol) and triethylamine (28.6 mg,0.283 mmol) at 0° C. The reaction mixture was stirred for 8 h at ambienttemperature. When the reaction was complete (monitored by TLC), waterwas added and the resulting aqueous mixture was extracted withdichloromethane. The organic layer was washed with water, followed bybrine solution, dried over anhydrous Na₂SO₄ and concentrated to affordthe crude product. The crude product was purified by columnchromatography (silica gel, 3% methanol in dichloromethane) to affordcompound JVM 3-36 as white solid (yield: 68%).

¹H NMR (CDCl₃, 400 MHz): δ 8.58 (s, 1H), 8.04 (d, J=7.6 Hz, 1H), 7.96(d, J=8.0 Hz, 1H), 7.90 (d, J=8.4 Hz, 2H), 7.63-7.54 (m, 2H), 6.23 (s,1H), 5.83 (t, J=8.4 Hz, 1H), 5.54 (s, 1H), 5.0 (d, J=12.8 Hz, 1H), 4.79(d, J=12.4 Hz, 1H), 3.89 (t, J=9.2 Hz, 1H), 3.05-2.99 (m, 1H), 2.94 (d,J=9.6 Hz, 1H), 2.55-2.17 (m, 7H), 1.73 (t, J=7.2 Hz, 111), 1.57 (s, 3H),1.15 (t, I=13.2 Hz, 1H).

REFERENCES FOR EXAMPLE 21-23

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What is claimed is:
 1. A method for inhibiting growth of a cancer cell,the method comprising contacting the cancer cell with an amount of acompound comprising Formula (I), or a salt thereof, effective to inhibitgrowth of the cancer cell, the compound comprising Formula (I):

wherein: R is selected from the group consisting of

wherein: R₁ is selected from the group consisting of substitutedindoles, substituted heterocyclic aromatic, and substituted aromaticderivatives; R₂ is selected from the group consisting of substitutedindoles; and R₃ is selected from the group consisting of substitutedamines; wherein the cancer cell is an acute myelogenous leukemia (AML)cancer cell or a solid tumor cell; and wherein the solid tumor cell isselected from the group consisting of non-small cell lung cancer, coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer and breast cancer.
 2. The method of claim 1, wherein Formula (I)comprises:

wherein: X is selected from the group consisting of S, O, NH, NR₁₀; R₉is selected from the group consisting of OMe, Cl, Br and F; and R₁₀ isselected from the group consisting of H, CH₃, benzyl, substitutedbenzyl, benzoyl, substituted benzoyl, and benzylsulfonyl and substitutedbenzylsulfonyl.
 3. The method of claim 1, wherein Formula (I) comprises:

Wherein: R₁₁ is selected from the group consisting of H, CH₃, benzyl,substituted benzyl, benzoyl, substituted benzoyl, and benzylsulfonyl andsubstituted benzylsulfonyl; and R₁₂ and R₁₃ are independently selectedfrom the group consisting of H, F, Cl, Br, OCH₃, CN, CH₃, NO₂ andCOOCH₃.
 4. The method of claim 1, wherein Formula (I) comprises:

wherein: R₁₄ is selected from the group consisting of 2-thiophenyl,3-thiophenyl, 2-pyrazine, 3-pyrazine, 2-amino nicotinic acid,indole-3-acetic acid, indole-3-acrylic acid.
 5. The method of claim 1,wherein Formula (I) comprises:

wherein: R₁₅ is selected from the group consisting of:


6. The method of claim 1, wherein Formula (I) comprises:

wherein: R₁ is selected from the group consisting of substitutedindoles, substituted heterocyclic aromatic, aromatic and aliphaticderivatives.
 7. The method of claim 1, wherein the cancer cell is invivo or in vitro.